Combination therapy

ABSTRACT

The present invention relates to a pharmaceutical combination comprising (a) a Raf inhibitor as defined herein, or a pharmaceutically acceptable salt thereof and (b) a MEK inhibitor, particularly trametinib, particularly for use in the treatment of a proliferative disease. This invention also relates to uses of such combination for preparation of a medicament for the treatment of a proliferative disease; methods of treating a proliferative disease in a subject in need thereof comprising administering to said subject a jointly therapeutically effective amount of said combination; use of such combination for the treatment of proliferative disease; pharmaceutical compositions comprising such combination and commercial packages thereto.

RELATED APPLICATIONS

This application is a U.S. National Phase filing of InternationalApplication Ser. No. PCT/IB2018/052989 filed 30 Apr. 2018 and claimspriority to U.S. Provisional Application Ser. No. 62/500,108 filed 2 May2017 all of which are incorporated in their entireties herein.

FIELD OF THE INVENTION

This invention relates to a pharmaceutical combination comprising (a) aRaf inhibitor selected from the group consisting of Compound of formula(I), as defined herein, or a pharmaceutically acceptable salt thereof,or Compound of formula (II), as defined herein, or a pharmaceuticallyacceptable salt thereof, and (b) a MEK inhibitor, particularlytrametinib, or a pharmaceutically acceptable salt or solvate thereof.This invention also relates to a pharmaceutical combination comprising(a) a Raf inhibitor selected from the group consisting of Compound offormula (I), as defined herein, or a pharmaceutically acceptable saltthereof, or Compound of formula (II), as defined herein, or apharmaceutically acceptable salt thereof, and trametinib, or apharmaceutically acceptable salt or solvate thereof.

This invention also relates to such combinations for use in thetreatment of a proliferative disease, in particular a cancer, uses ofsuch combinations for the preparation of a medicament for the treatmentof a proliferative disease, in particular a cancer; methods of treatinga proliferative disease, in particular a cancer, in a subject in needthereof comprising administering to said subject a jointlytherapeutically effective amount of said combinations; use of suchcombinations for the treatment of a proliferative disease, in particulara cancer; pharmaceutical compositions comprising such combinations andcommercial packages thereto.

This invention also relates to Compound of formula (I), as definedherein, or a pharmaceutically acceptable salt thereof, or Compound offormula (II), as defined herein, or a pharmaceutically acceptable saltthereof, for use in a combination therapy with a MEK inhibitor,particularly trametinib, or a pharmaceutically acceptable salt orsolvate thereof. There is also provided herein a MEK inhibitor,particularly trametinib, or a pharmaceutically acceptable salt orsolvate thereof, for use in a combination therapy with Compound offormula (I), as defined herein, or a pharmaceutically acceptable saltthereof, or for use in a combination therapy with Compound of formula(II), as defined herein, or a pharmaceutically acceptable salt thereof.

BACKGROUND

The RAS/RAF/MEK/ERK or MAPK pathway is a key signaling cascade thatdrives cell proliferation, differentiation, and survival. Dysregulationof this pathway underlies many instances of tumorigenesis. This pathwayis activated by extracellular signals that in turn induces the small Gprotein RAS to exchange GDP for GTP. The activated RAS small guanidinetriphosphatase (GTPase) promotes the activation of the RAF (alsoreferred to as “Raf” herein) family proteins (ARAF, BRAF and CRAF, alsoknown as RAF1). Activated RAF proteins lead to the phosphorylation andactivation of MEK1/2 proteins, which subsequently phosphorylate andactivate extracellular signal-regulated kinases (ERKs). ERK1/2 proteinsphosphorylate a variety of substrates, including multiple transcriptionfactors, and regulate key cellular activities, including proliferation,differentiation, migration, survival and angiogenesis.

Aberrant signaling or inappropriate activation of the MAPK pathway hasbeen shown in multiple tumor types, including melanoma, lung andpancreatic cancer, and can occur through several distinct mechanisms,including activating mutations in RAS and BRAF (V-Raf Murine SarcomaViral Oncogene Homolog B1). RAS which is a superfamily of GTPasesincludes KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog),which is a regulated signaling protein that can be turned on (activated)by various single-point mutations, which are known as gain-of-functionmutations. RAS mutations, particularly gain-of-function (GOF) mutations,have been detected in 9-30% of all cancers, with KRAS mutations havingthe highest prevalence (86%), followed by NRAS (11%), and, infrequently,HRAS (3%) (Cox AD, et al, Nat Rev Drug Discov 2014;13(11):828-51).Activating KRAS mutations are also frequently found in melanoma(Fedorenko IV, et al, Br J Cancer 2015;112(2):217-26), pancreatic cancer(di Magliano MP & Logsdon CD, Gastroenterology 2013;144(6):1220-9),colorectal cancer (Knickelbein K & Zhang L, Genes Dis 2015;2(1):4-12)and ovarian cancer (Nakayama N, et al, Br J Cancer 2008;99(12):2020-8).

Inhibitors that target downstream effectors of RAS, such as RAF, MEK,and ERK kinases, have not demonstrated significant clinical activtiy inRAS-driven tumors. For example, RAF inhibitors, such as vemurafenibwhich are efficacious in BRAF v⁶⁰⁰-mutant melanomas, are ineffective inRAS mutant cancers. There are thus currently no effective therapies forKRAS-mutant tumors and NRAS-mutant tumors. In particular, unlike BRAFmutant melanoma, there are no approved targeted therapies for NRASmutant melanoma patients. Recent data from a trial of a MEK1/2 inhibitordemonstrated a small increase in progression free survival but noimprovement in overall survival in these patients (Dummer et al., LancetOncol, 18, 435-445, 2017).

Although some vertical combinations of MAPK inhibitors have provedbeneficial, it is not always predictable whether any one of the variouspermutations of possible combinations would be of clinical benefit. Forexample, it was recently reported that a combination of the MEKinhibitor cobimetinib in combination with the ERK1/2 inhibitor GDC-0994led to overlapping and cumulative toxicity, which, according to theauthors, would restrict further development of this particularcombination. (Weekes et al 2017, Abstract CT107:AACR Annual Meeting2017; Apr. 1-5, 2017 Combinations)

Lung cancer is a common type of cancer that affects men and women aroundthe globe. NSCLC is the most common type (roughly 85%) of lung cancerwith approximately 70% of these patients presenting with advanceddisease (Stage IIIB or Stage IV) at the time of diagnosis. About 30% ofNSCLC tumors contain activating KRAS mutations, and these mutations areassociated with resistance to epidermal growth factor receptor tyrosinekinase inhibitors (EGFR TKIs) (Pao W, et al, PLoS Med 2005;2(1):e17). Todate, no approved targeted therapies are available for patientssuffering from NSCLC with KRAS mutation and from NSCLC with BRAFmutations other than V600E.

Melanoma is a common type of cancer that affects men and women aroundthe globe. 50% of metastatic cutaneous melanoma patients harbor aBRAF-activating mutation and 20% of these patients harbour anNRAS-activating mutation (Zhang et al, Pigment Cell Melanoma Res 2016;29:266-283. NRAS mutations were identified as an independent predictorof shorter survival after a diagnosis of stage IV melanoma (Jakob JA etal (2012), Cancer, Volume 118, Issue 16, Pages 4014-4023).

Direct inhibition of KRAS and NRAS is thus still proving challenging andto date no approved targeted therapies are available for patients withKRAS-mutant cancers, such as KRAS mutant NSCLC, and NRAS-mutant cancers,such as NRAS mutant melanoma. Thus, there is a need for targeted therapythat is safe, is well tolerated, and/or is accompanied with feweradverse side-effects such as skin rash. A therapy which results indurable and sustained responses in a clinical setting is also needed.

SUMMARY

It has now been found that that a combination of a MEK inhitbitor and aselective Raf inhibitor, such as the Compound of formula (I) as definedherein, that potently inhibit the activity of both CRAF and BRAF may beeffective in blocking BRAF-mutant tumors and RAS-mutant driventumorigenesis. Combining Compound of formula (I) and trametinib wasfound to be synergistic in MAPK-mutant cancer cell lines suchNRAS-mutant and KRAS-mutant cell lines cell lines. Based on the findingsdescribed herein, a combination of a Raf inhibitor, particularly a CRAF-and BRAF-inhibitor such as a Compound of formula (I), with a MEKinhibitor, such as trametinib, may be particularly efficacious and lesssusceptible to resistance in patients suffering from RAS-mutant tumors.

The combination of Compound of formula (I) with trametinib alsodemonstrated increased anti-tumor response compared to eithersingle-agent therapy in human KRAS mutant NSCLC, CRC, and PDAC xenograftmodels, and human NRAS mutant melanoma xenograft models. The combinationof Compound of formula (II) with trametinib demonstrated increasedefficacy of tumor response compared to either single-agent therapy in ahuman HPAFII pancreatic xenograft mouse model. Therefore, Compound offormula (I) or Compound of formula (II), alone and in combination with aMEK inhibitor, may be useful in the treatment of patients with cancersharboring MAPK pathway alterations. Such cancers include KRAS-mutantNSCLC (non-small cell lung cancer), KRAS-mutant pancreatic cancer (e.g.KRAS-mutant pancreatic ductal adenocarcinoma (PDAC), KRAS-mutant CRC(colorectal cancer), and NRAS-mutant melanoma).

The present invention thus provides a pharmaceutical combinationcomprising (a) a Raf inhibitor selected from the group consisting of (i)Compound of formula (I), as defined herein, or a pharmaceuticallyacceptable salt thereof, and (ii) Compound of formula (II), as definedherein, or a pharmaceutically acceptable salt thereof, and (b) a MEKinhibitor, particularly trametinib, or a pharmaceutically acceptablesalt or solvate thereof The present invention also provides such acombination for use in the treatment of a proliferative disease.

In addition, the present invention provides:

(a) a Raf inhibitor selected from the group consisting of (i) Compoundof formula (I), as defined herein, or a pharmaceutically acceptable saltthereof, and (ii) Compound of formula (II), as defined herein, or apharmaceutically acceptable salt thereof, for use in a combinationtherapy with a MEK inhibitor, particularly trametinib, or apharmaceutically acceptable salt or solvate thereof; and

(b) a MEK inhibitor, particularly trametinib, or a pharmaceuticallyacceptable salt or solvate thereof, for use in a combination therapywith a Raf inhibitor selected from the group consisting of (i) Compoundof formula (I), as defined herein, or a pharmaceutically acceptable saltthereof, and (ii) Compound of formula (II), as defined herein, or apharmaceutically acceptable salt thereof.

Compound of formula (I) is the compound with the following structure:

Compound of formula (II) is the compound with the following structure:

The present invention further provides a pharmaceutical combinationcomprising a Raf inhibitor compound, as defined herein, or apharmaceutically acceptable salt thereof, and a MEK inhibitor,preferably trametinib, or a pharmaceutically acceptable salt or solventthereof, particularly for simultaneous, separate or sequential use inthe treatment of a proliferative disease.

In another preferred embodiment, the pharmaceutical combination of thepresent invention comprises (a) a Raf inhibitor Compound of formula (I),or a pharmaceutically acceptable salt thereof, and (b) trametinib, or apharmaceutically acceptable salt or solvate thereof, in particular asolvate thereof.

In another preferred embodiment, the pharmaceutical combination of thepresent invention comprises (a) a Raf inhibitor Compound of formula(II), or a pharmaceutically acceptable salt thereof, and (b) trametinib,or a pharmaceutically acceptable salt or solvate thereof, in particulara solvate thereof.

The present invention is particularly related to the combination of theinvention for use in the treatment of a cancer characterized byactivating mutations in the MAPK pathway, and in particular by one ormore mutations in RAS (e.g. KRAS or NRAS) and/or BRAF.

The present invention also provides the use of the combination of theinvention for the treatment of a proliferative disease, particularly acancer. In particular, the combination of the invention may be usefulfor the treatment of non-small cell lung cancer (NSCLC), melanoma,pancreatic ductal adenocarcinoma (PDAC), cervical cancer, ovarian canceror colorectal cancer (CRC).

The present invention also provides the use of the combination of theinvention for the preparation of a medicament for the treatment of aproliferative disease, particularly a cancer.

The present invention also provides a method of treating a proliferativedisease comprising simultaneously, separately or sequentiallyadministering to a subject in need thereof a combination of theinvention in a quantity which is jointly therapeutically effectiveagainst said proliferative disease.

The present invention also provides a pharmaceutical composition orcombined preparation comprising a quantity of the combination of theinvention, which is jointly therapeutically effective against aproliferative disease, and optionally at least one pharmaceuticallyacceptable carrier.

The present invention also provides a combined preparation comprising(a) one or more dosage units of a Raf inhibitor selected from the groupconsisting of (i) Compound of formula (I), or a pharmaceuticallyacceptable salt thereof, and (ii) Compound of formula (II), or apharmaceutically acceptable salt thereof, and (b) one or more dosageunits of a MEK inhibitor, preferably trametinib, or a pharmaceuticallyacceptable salt or solvate thereof, for use in the treatment of aproliferative disease.

The present invention also provides a commercial package comprising asactive ingredients a combination of the invention and instructions forsimultaneous, separate or sequential administration of a combination ofthe invention to a patient in need thereof for use in the treatment of aproliferative disease, preferably non-small cell lung cancer (NSCLC),melanoma, pancreatic ductal adenocarcinoma (PDAC), cervical cancer,ovarian cancer or colorectal cancer (CRC).

The present invention also provides a commercial package comprising aRaf inhibitor selected from the group consisting of (i) Compound offormula (I), or a pharmaceutically acceptable salt thereof, and (ii)Compound of formula (ii), or a pharmaceutically acceptable salt thereof,and instructions for the simultaneous, separate or sequential use with aMEK inhibitor, preferably trametinib, or a pharmaceutically acceptablesalt or solvate thereof, in the treatment of a proliferative disease.

Various aspects of the invention are described in further detail below.Additional definitions are set out throughout the specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4. Compound of formula (I) in FIG. 1) Calu-6 (KRAS(Q61K)mutant), FIG. 2) NCI-H358 (KRAS(G12C) mutant), FIG. 3) HLUX1156(KRAS(G12C) mutant) and FIG. 4) NCI-H727 (KRAS(G12V) mutant) xenograftlung cancer tumor models in mice. Animals with subcutaneous xenograftsreceived treatment with Compound of formula (I) as indicated. Compoundwas administered orally (PO) either daily (qd or QD), twice daily (bidor BID) or every other day (q2d or Q2D) as indicated. Anti-tumoractivity was determined by assessing percentage of tumor volume in thetreatment groups versus that in vehicle-treated group (% T/C) orpercentage of tumor regression (i.e., volume of tumor after treatmentcompared to the starting volume (% regression) (indicated as “% Reg” or“−% Reg” in FIGS. 1-4). SEM=standard error of the mean, PBS=phosphatebuffered saline.

FIGS. 5 and 6 depict efficacy of Compound of formula (I) and MEKinhibitor trametinib used in combination in Calu-6 xenograft tumormodels in mice. Animals with subcutaneous xenografts of Calu-6 receivedtreatment with Compound of formula (I) and/or trametinib as indicated.Both depth (FIG. 5) and durability of response (FIG. 6) weredemonstrated. Compounds were administered orally either daily (qd) orevery other day (q2d) as indicated. Anti-tumor activity was determinedby assessing percentage of tumor volume in the treatment groups versusthat in vehicle-treated group (% T/C) or percentage of tumor regressioncompared to the starting volume (% regression, indicated as “% Reg” inFIGS. 2A-2B).

FIG. 7. Cell lines harboring different BRAF or RAS mutations weretreated with DMSO, dabrafenib or Compound of formula (II) at theindicated concentrations for 2 hr. Inhibition of MEK or ERKphosphorylation was measured by western blot analysis. Compound offormula (II) inhibits oncogenic signaling and proliferation in tumorcells with BRAF, NRAS or KRAS mutations with minimal paradoxicalactivation.

FIG. 8. Growth inhibition of cell lines after 5 days of treatment withdabrafenib (upper panel) or Compound of formula (II) (lower panel) wasdetermined.

FIG. 9. Dot plots of IC₅₀ values for growth inhibition in 357 humancancer cell lines by Compound of formula (II) or dabrafenib following 3days of inhibitor treatment. The dotted line represents an IC₅₀ of 5μMwhich was used as a cut-off for cell line sensitivity to the inhibitors.The number of sensitive and resistant cell lines to each inhibitor amongBRAF mutant, KRAS mutant, NRAS mutant, or wild-type (WT) cells isindicated below the graph. A Fisher's Exact Test was performed todetermine the statistical significance of inhibitor activity in BRAF orRAS mutant cell lines vs WT cell lines.

FIG. 10. Tumor samples were collected at the indicated time pointsfollowing a single dose of vehicle or increasing doses of Compound offormula (II) in the Calu-6 tumor-bearing animals to determinephosphorylated MEK (pMEK) levels. pMEK levels are represented as theratio of pMEK/total MEK in the treatment group compared to vehiclecontrol at each time point.

FIG. 11. Calu-6 tumor xenograft growth inhibition was measured followingtreatment with vehicle or Compound of formula (II) across four doselevels. Tumor volume is represented as the mean tumor volume from 6animals per treatment group±standard error of the mean (SEM). Animalswith subcutaneous xenografts of Calu-6 received treatment with Compoundof formula (II) as indicated. Compound of formula (II) was administereddaily (qd). Anti-tumor activity was determined by assessing percentageof tumor volume in the treatment groups versus that in thevehicle-treated group (% T/C) or percentage of tumor regression comparedto the starting volume (% regression, indicated as “% Reg” in FIG. 11).

FIG. 12. In vivo antitumor activity of Compound of formula (II) wasassessed in a panel of 23 NSCLC patient derived xenograft (PDX) models,presented as the % of change in tumor volume at the time of measurementcompared to initial tumor volume, positive values indicated tumor growthand negative values indicated tumor regression. Compound of formula (II)was dosed orally once daily at 60 mg/kg or 200 mg/kg (indicated by ▾).Each tumor is annotated for BRAF or RAS mutation status. Tumor responseto paclitaxel in a panel of NSCLC PDX models was included forcomparison. GOF=gain-of-function, WT=wildtype.

FIG. 13. Anti-proliferative combination activity of Compound of formula(II) and trametinib in HPAF-II (KRAS(G12D mutant) pancreas derived cellswas assessed. Top left panel: dose matrix representing percentages ofgrowth inhibition relative to DMSO by Compound of formula (II),trametinib, and the combination following 5 days of treatment. Top rightpanel: excess inhibition values representing the deviation between thecombination effect and the calculated additivity effect of the twosingle agents using the Loewe model. The calculated Loewe synergy scoreis indicated. Lower panel: isobologram analysis of the dose matrix data,the dark grey line represents the data points and the light grey lineindicates additivity. The calculated Loewe Combination Index (CI) at 50%growth inhibition is indicated.

FIG. 14. In vivo activity of Compound of formula (II) and trametinib assingle agents or in combination in the HPAF-II xenograft model.Signaling inhibition following a single dose of the treatment asmeasured by DUSP6 (dual specificity phosphatase 6) mRNA levels. Inaddition, an efficacious dosage can be determined by monitoringbiomarkers indicative of MAP kinase pathway inhibition. In particular,DUSP6 is a known biomarker for this pathway, and in vivo levels of DUSP6have been shown to drop in response to Compound of formula (II) that isassociated with efficacious plasma levels of Compound of formula (II).

DUSP6 levels are represented as the percentage change in comparison tothe vehicle group after normalization to control gene RPLPO.

FIG. 15. In vivo activity of Compound of formula (II) and trametinib assingle agents or in combination in the HPAF-II xenograft model. In vivotumor growth following ten days of treatment as indicated. Compound wasadministered daily (qd). Anti-tumor activity was determined by assessingpercentage of tumor volume in the treatment groups versus that invehicle-treated (% T/C) or percentage of tumor regression compared tothe starting volume (% regression, indicated as “% Reg” in FIG. 15).

FIG. 16. A. In vivo activity of Compound of formula (I) (Compound I inFigure) and trametinib as single agents or in combination in HCT116xenograft model. In vivo tumor growth following seventeen days oftreatment as indicated. Compounds were administered daily (qd) or everyother day (q2d). Anti-tumor activity was determined by assessingpercentage of tumor volume in the treatment groups versus that invehicle-treated (% T/C) or percentage of tumor regression compared tothe starting volume (% regression, indicated as “% Reg” in FIG. 16).

FIG. 17. A. In vivo activity of Compound of formula (I) (Compound I inFigure) and trametinib as single agents or in combination in 2043xenograft model. In vivo tumor growth following 21 days of treatment asindicated. Compounds were administered daily (qd) or every other day(q2d). Anti-tumor activity was determined by assessing percentage oftumor volume in the treatment groups versus that in vehicle-treated (%T/C) or percentage of tumor regression compared to the starting volume(% regression, indicated as “% Reg” in FIG. 17).

FIG. 18. A. In vivo activity of Compound of formula (I) (Compound I inFigure) and trametinib as single agents or in combination in SKMEL30xenograft model. In vivo tumor growth following 22 days of treatment asindicated. Compounds were administered daily (qd) or twice daily (bid).Anti-tumor activity was determined by assessing percentage of tumorvolume in the treatment groups versus that in vehicle-treated (% T/C) orpercentage of tumor regression compared to the starting volume (%regression, indicated as “% Reg” in FIG. 18).

FIG. 19. A. In vivo activity of Compound of formula (I) (Compound I inFigure) and trametinib as single agents or in combination in 20667xenograft model. In vivo tumor growth following 17 days of treatment(single agents) and 31 days of combination treatment as indicated.Compounds were administered daily (qd) or twice daily (bid). Anti-tumoractivity was determined by assessing percentage of tumor volume in thetreatment groups versus that in vehicle-treated (% T/C) on day 34 forsingle agents or percentage of tumor regression compared to the startingvolume (% regression, indicated as “% Reg” in FIG. 19) on day 48 for thecombination.

FIG. 20. A. In vivo activity of Compound of formula (I) (Compound I inFigure) and trametinib as single agents or in combination in 21124xenograft model. In vivo tumor growth following 21 days of treatment asindicated. Compounds were administered daily (qd) or twice daily (bid).Anti-tumor activity was determined by assessing percentage of tumorvolume in the treatment groups versus that in vehicle-treated (% T/C) orpercentage of tumor regression compared to the starting volume (%regression, indicated as “% Reg” in FIG. 20).

FIG. 21. A. In vivo activity of Compound of formula (I) (Compound I inFigure) and trametinib as single agents or in combination in 20864xenograft model. In vivo tumor growth following 14 days of treatment(single agents) and 36 days of combination treatment as indicated.Compounds were administered daily (qd) or twice daily (bid). Anti-tumoractivity was determined by assessing percentage of tumor volume in thetreatment groups versus that in vehicle-treated (% T/C) on day 33 forsingle agents or percentage of tumor regression compared to the startingvolume (% regression, indicated as “% Reg” in FIG. 21) on day 55 for thecombination.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a pharmaceutical combination comprising(a) a Raf inhibitor selected from the group consisting of (i) Compoundof formula (I), as defined herein, or a pharmaceutically acceptable saltthereof, and (ii) Compound of formula (II), as defined herein, or apharmaceutically acceptable salt thereof, and (b) a MEK inhibitor,particularly for use in the treatment of a proliferative disease.

As used herein, the term “Raf inhibitor” refers to an adenosinetriphosphate (ATP)-competitive inhibitor of B-Raf protein kinase (alsoreferred to herein as b-RAF, BRAF or b-Raf) and C-Raf protein kinase(also referred to herein as c-RAF, CRAF or c-Raf) that selectivelytargets, decreases, or inhibits at least one activity ofserine/threonine-protein kinase B-Raf or C-Raf. The Raf inhibitorpreferentially inhibits both Raf monomers and Raf dimers.

As used herein, the Raf inhibitor is selected from the group consistingof (i) Compound of formula (I), as defined herein, or a pharmaceuticallyacceptable salt thereof, and (ii) Compound of formula (II), as definedherein, or a pharmaceutically acceptable salt thereof.

Compound of formula (I) has the following structure:

For convenience, the group of the compound and its salts is collectivelyreferred to as “Compound of formula (I)” or “Compound (I)”, meaning thatreference to “Compound of formula (I)” or “Compound (I)” will refer toany of the compound or pharmaceutically acceptable salt thereof in thealternative.

Raf inhibitor Compound of formula (I) and its pharmaceuticallyacceptable salts are described in WO2014/151616, which is herebyincorporated by reference in its entirety, and methods of itspreparation have been described, for example, in Example 1156 therein.

Compound of formula (II) has the following structure:

For convenience, the group of the compound and its salts is collectivelyreferred to as “Compound of formula (II)” or “Compound (II)”, meaningthat reference to “Compound of formula (II)” or “Compound (II)” willrefer to any of the compound or pharmaceutically acceptable salt thereofin the alternative.

Raf inhibitor Compound of formula (II) and its pharmaceuticallyacceptable salts are described in WO2014/151616, which is herebyincorporated by reference in its entirety, and methods of itspreparation have been described, for example, in Example 131 therein.

In cell-based assays, Raf inhibitors Compound of formula (I) andCompound of formula (II) demonstrated anti-proliferative activity incell lines that contain a variety of mutations that activate MAPKsignaling. In vivo, treatment with Compound of formula (I) or Compoundof formula (II) generated tumor regression in several KRAS-mutant modelsincluding the NSCLC-derived Calu-6 (KRAS Q61K) and NCI-H358 (KRAS G12C).Collectively, in vitro and in vivo MAPK-pathway suppression andanti-proliferative activity observed for Compound of formula (I) orCompound of formula (II) at well-tolerated doses suggest that Compoundof formula (I) or Compound of formula (II) may have anti-tumor activityin patients with tumors harboring activating lesions in the MAPKpathway. Moreover, Compound of formula (I) and Compound of formula (II)are a Type 2 ATP-competitive inhibitor of both B-Raf and C-Raf thatkeeps the kinase pocket in an inactive conformation, thereby reducingthe paradoxical activation seen with many B-Raf inhibitors, and blockingmutant Ras-driven signaling and cell proliferation. Compound of formula(I) and Compound of formula (II) exhibited efficacy in numerousMAPK-driven human cancer cell lines and in xenograft tumors representingmodel tumors harboring human lesions in KRAS, NRAS and BRAF oncogenes.

Pharmaceutical combinations of the present invention further comprise aMEK inhibitor. The term “MEK inhibitor” is defined herein to refer to acompound which targets, decreases or inhibits at least one activity ofMAP/ERK kinases 1 and 2 (MEK1/2).

Suitable MEK inhibitors for use in the combination of the presentinvention include, but are not limited to

-   -   a) trametinib        (N-(3-{3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydropyrido[4,3-d]pyrimidin-1(2H)-yl}phenypacetamide),        also referred to as JPT-74057 or GSK1120212, or a        pharmaceutically acceptable salt or solvate thereof trametinib        is disclosed in Example 4-1 in PCT Publication No. WO        2005/121142, which is hereby incorporated by reference in its        entirety. As a monotherapy trametinib has been approved for the        treatment of unresectable or metastatic malignant melanoma with        B-Raf V600E or V600K mutations and the compound is commercially        available from Novartis AG under the trade name Mekinist®.    -   b) PD0325901 (Pfizer) (disclosed in PCT Publication No.        WO02/06213); PD184352 (Pfizer); Refametinib (also referred to as        RDEA119 or Bay 86-9766); Cobimetinib, also referred to as XL518,        and commercially available from Roche under the trade name        Cotellic®; AS-701255 (Merck Serono); AS-701173 (Merck Serono);        Pimasertib, also referred to as AS-703026 or MSC1936369B (Merck        Serono); RDEA436 (Ardea Biosciences); RO4987655, also referred        to as RG 7167 (Roche) and/or RG7420, also referred to as        GDC-0623 (Roche), or a pharmaceutically acceptable salt thereof.

Preferably, the MEK inhibitor is trametinib, or a pharmaceuticallyacceptable salt or solvate thereof In some preferred embodiments,trametinib is in the form of a dimethyl sulfoxide solvate. In someembodiments, trametinib is in the form of a sodium salt. Suitably,trametinib is in the form of a solvate selected from: hydrate, aceticacid, ethanol, nitromethane, chlorobenzene, 1-pentancol, isopropylalcohol, ethylene glycol and 3-methyl-1-butanol. These solvates and saltforms can be prepared by one of skill in the art from the description inWO 2005/121142.

The present invention further relates to a pharmaceutical combinationcomprising (a) a Raf inhibitor selected from the group consisting of (i)Compound of formula (I), as defined herein, or a pharmaceuticallyacceptable salt thereof, and (ii) Compound of formula (II), as definedherein, or a pharmaceutically acceptable salt thereof and (b) a MEKinhibitor, particularly for simultaneous, separate or sequential use inthe treatment of a proliferative disease.

Selected terms are defined below and throughout the application.Compounds of the present invention are described using standardnomenclature. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as is commonly understood by oneof skill in the art to which this invention belongs. The followinggeneral definitions shall apply in this specification, unless otherwisespecified:

As used herein the term “combination of the invention” refers to thecombined administration comprised of (a) a Raf inhibitor selected fromthe group consisting of (i) Compound of formula (I), as defined herein,or a pharmaceutically acceptable salt thereof, and (ii) Compound offormula (II), as defined herein, or a pharmaceutically acceptable saltthereof, and (b) a MEK inhibitor, preferably trametinib, or apharmaceutically acceptable salt or solvate thereof. The Raf inhibitorCompound of formula (I), or a pharmaceutically acceptable salt thereof,and the MEK inhibitor, preferably trametinib, or a pharmaceuticallyacceptable salt or solvate thereof, or the Raf inhibitor Compound offormula (II), or a pharmaceutically acceptable salt thereof, and the MEKinhibitor, preferably trametinib, or a pharmaceutically acceptable saltor solvate thereof, may be employed in combination in accordance withthe invention by administration simultaneously in a unitarypharmaceutical composition including both compounds. Alternatively, thecombination may be administered separately in separate pharmaceuticalcompositions, each including one of the Raf inhibitors and a MEKinhibitor in a sequential manner wherein, for example, the Raf inhibitoror a MEK inhibitor is administered first and the other second. Suchsequential administration may be close in time (e.g., simultaneously) orremote in time.

As used herein, the terms “a” and “an” and “the” and similar referencesin the context of describing the invention are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. Where the plural form is used forcompounds, salts, and the like, this is taken to mean also a singlecompound, salt, or the like.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values. When describing a dosage herein as “about” a specifiedamount, the actual dosage can vary by up to 10% from the stated amount:this usage of “about” recognizes that the precise amount in a givendosage form may differ slightly from an intended amount for variousreasons without materially affecting the in vivo effect of theadministered compound.

When describing a dosage herein as a specified amount, i.e. without theterm “about”, the actual dosage can vary by up to 10% (preferably by upto 5%) from the stated amount: this usage recognizes that the preciseamount in a given dosage form may differ slightly from an intendedamount for various reasons without materially affecting the in vivoeffect of the administered compound.

The terms “comprising” and “including” are used herein in theiropen-ended and non-limiting sense unless otherwise noted.

By “a combination” or “in combination with” it is not intended to implythat the therapy or the therapeutic agents must be physically mixed oradministered at the same time and/or formulated for delivery together,although these methods of delivery are within the scope describedherein. A therapeutic agent in these combinations can be administeredconcurrently with, prior to, or subsequent to, one or more otheradditional therapies or therapeutic agents. The therapeutic agents canbe administered in any order. In general, each agent will beadministered at a dose and/or on a time schedule determined for thatagent. It will further be appreciated that the additional therapeuticagent utilized in this combination may be administered together in asingle composition or administered separately in different compositions.In general, it is expected that additional therapeutic agents utilizedin combination be utilized at levels that do not exceed the levels atwhich they are utilized individually. In some embodiments, the levelsutilized in combination will be lower than those utilized assingle-agent therapeutics.

The combinations of the invention have therapeutic or protectivefunctions or both. For example, these molecules may be administered to ahuman subject, to treat and/or prevent a variety of disorders, such ascancers as described herein.

The terms “combination”, “therapeutic combination” or “pharmaceuticalcombination” as used herein refer to either a fixed combination in onedosage unit form, or non-fixed combination, or a kit of parts for thecombined administration where two or more therapeutic agents may beadministered together, independently at the same time or separatelywithin time intervals, especially where these time intervals allow thatthe combination partners show a cooperative, e.g., synergistic, effect.

The term “combination therapy” refers to the administration of two ormore therapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single formulation having a fixedratio of active ingredients or in separate formulations (e.g., capsulesand/or intravenous formulations) for each active ingredient. Inaddition, such administration also encompasses use of each type oftherapeutic agent in a sequential or separate manner, either atapproximately the same time or at different times. Regardless of whetherthe active ingredients are administered as a single formulation or inseparate formulations, the drugs are administered to the same patient aspart of the same course of therapy. In any case, the treatment regimenwill provide beneficial effects in treating the conditions or disordersdescribed herein.

By simultaneous therapeutic use, within the meaning of the presentinvention is meant an administration of at least two active ingredientsby the same route and at the same time or at substantially the sametime.

By separate use, within the meaning of the present invention is meant inparticular an administration of at least two active ingredients at thesame time or at substantially the same time by different routes.

By sequential therapeutic use is meant administration of at least twoactive ingredients at different times, the administration route beingidentical or different. More particularly by an administration method ismeant according to which the whole administration of one of the activeingredients is carried out before administration of the other or otherscommences.

The terms “fixed combination”, “fixed dose” and “single formulation” asused herein refers to a single carrier or vehicle or dosage formformulated to deliver an amount, which is jointly therapeuticallyeffective for the treatment of cancer, of both therapeutic agents to apatient. The single vehicle is designed to deliver an amount of each ofthe agents along with any pharmaceutically acceptable carriers orexcipients. In some embodiments, the vehicle is a tablet, capsule, pill,or a patch. In other embodiments, the vehicle is a solution or asuspension.

The term “non-fixed combination” or “kit of parts” means that thetherapeutic agents of the combination of the invention are bothadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific time limits, wherein suchadministration provides therapeutically effective levels of the twocompounds in the body of a subject in need thereof. The latter alsoapplies to cocktail therapy, e.g., the administration of three or moreactive ingredients.

The term “pharmaceutically acceptable” as used herein refers to thosecompounds, materials, compositions and/or dosage forms, which are,within the scope of sound medical judgment, suitable for contact withthe tissues of a subject, e.g., a mammal or human, without excessivetoxicity, irritation, allergic response and other problems orcomplications commensurate with a reasonable benefit/risk ratio.

As used herein, the term “pharmaceutically acceptable excipient” or“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, surfactants, antioxidants, preservatives(e.g., antibacterial agents, antifungal agents), isotonic agents,absorption delaying agents, salts, preservatives, drugs, drugstabilizers, binders, excipients, disintegration agents, lubricants,sweetening agents, flavoring agents, dyes, and the like and combinationsthereof, as would be known to those skilled in the art. Except insofaras any conventional carrier is incompatible with the active ingredient,its use in the therapeutic or pharmaceutical compositions iscontemplated.

The term “pharmaceutical composition” is defined herein to refer to amixture or solution containing at least one therapeutic agent to beadministered to a subject, e.g., a mammal or human, in order or treat aparticular disease or condition affecting the subject. The presentpharmaceutical combinations can be formulated in suitable pharmaceuticalcompositions for enteral or parenteral administration, such assugar-coated tablets, tablets, capsules or suppositories, or ampoules.If not indicated otherwise, these are prepared in a manner known per se,for example by means of various conventional mixing, comminution, directcompression, granulating, sugar-coating, dissolving, lyophilizingprocesses, or fabrication techniques readily apparent to those skilledin the art. It will be appreciated that the unit content of acombination partner contained in an individual dose of each dosage formneed not in itself constitute an effective amount since the necessaryeffective amount may be reached by administration of a plurality ofdosage units. The pharmaceutical composition may contain, from about0.1% to about 99.9%, preferably from about 1% to about 60%, of thetherapeutic agent(s). One of ordinary skill in the art may select one ormore of the aforementioned carriers with respect to the particulardesired properties of the dosage form by routine experimentation andwithout any undue burden. The amount of each carriers used may varywithin ranges conventional in the art. The following references disclosetechniques and excipients used to formulate oral dosage forms: TheHandbook of Pharmaceutical Excipients, 4th edition, Rowe et al., Eds.,American Pharmaceuticals Association (2003); and Remington: the Scienceand Practice of Pharmacy, 20th edition, Gennaro, Ed., LippincottWilliams & Wilkins (2003). These optional additional conventionalcarriers may be incorporated into the oral dosage form either byincorporating the one or more conventional carriers into the initialmixture before or during granulation or by combining one or moreconventional carriers with granules comprising the combination of agentsor individual agents of the combination of agents in the oral dosageform. In the latter embodiment, the combined mixture may be furtherblended, e.g., through a V-blender, and subsequently compressed ormolded into a tablet, for example a monolithic tablet, encapsulated by acapsule, or filled into a sachet.

Pharmaceutical compositions may be presented in unit dose formscontaining a predetermined amount of active ingredient per unit dose. Incertain embodiments, the unit dose includes one or more vehicles suchthat each vehicle includes an effective amount of at least one of thetherapeutic agents along with pharmaceutically acceptable carriers andexcipients. In some embodiments, the unit dose is one or more tablets,capsules, pills, injections, infusions, patches, or the like,administered to the patient at the same time. As is known to thoseskilled in the art, the amount of active ingredient per dose will dependon the condition being treated, the route of administration and the age,weight and condition of the patient. Preferred unit dosage compositionsare those containing a daily dose or sub-dose, or an appropriatefraction thereof, of an active ingredient. Furthermore, suchpharmaceutical compositions may be prepared by any of the methods wellknown in the pharmacy art.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or “effective amount” of a compoundof the invention. The term “pharmaceutically effective amount”,“therapeutically effective amount” or “clinically effective amount” of acombination of therapeutic agents is an amount sufficient, at dosagesand for periods of time necessary, to provide an observable orclinically significant improvement over the baseline of clinicallyobservable signs and symptoms of the disorders treated with thecombination. A therapeutically effective amount may vary according tofactors such as the disease state, age, sex, and weight of theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the therapeutic agents are outweighed bytherapeutically beneficial effects. A “therapeutically effective dosage”preferably modulates a measurable parameter, such as tumor growth rateor disease progression in a desired manner. The ability of a compound tomodulate a measurable parameter can be evaluated in an animal modelsystem predictive of efficacy in human tumors to help establish suitabledosing levels and schedules. Alternatively, this property of acomposition can be evaluated by examining the ability of the compound tomodulate an undesired parameter by using in vitro assays known to theskilled practitioner.

The term “jointly therapeutically active” or “joint therapeutic effect”as used herein means that the therapeutic agents can be given jointly,separately or sequentially in such time intervals that they prefer suchthat the subject, especially human, to be treated, still show an(preferably synergistic) interaction (joint therapeutic effect). Whetherthis is the case can, inter alia, be determined by following the bloodlevels of the compounds, showing that both compounds are present in theblood of the human to be treated at least during certain time intervals.

As used herein the term “agent” is understood to mean a substance thatproduces a desired effect in a tissue, system, animal, mammal, human, orother subject. It is also to be understood that an “agent” may be asingle compound or a combination or composition of two or morecompounds.

The term “proliferative disease” is preferably a cancer.

As used herein, the term “cancer” refers to a disease characterized bythe undesired and uncontrolled growth of aberrant cells. Cancer cellscan spread locally or through the bloodstream and lymphatic system toother parts of the body. As used herein, the term “cancer” or “tumor”includes premalignant, as well as malignant cancers and tumors. The term“cancer” is used herein to mean a broad spectrum of tumors, includingall solid and hematological malignancies.

An “oral dosage form” includes a unit dosage form prescribed or intendedfor oral administration.

As used herein, the terms “treat”, “treatment” and “treating” refer tothe reduction or amelioration of the progression, severity and/orduration of a disorder, e.g., a proliferative disorder, or theamelioration of one or more symptoms, suitably of one or morediscernible symptoms, of the disorder resulting from the administrationof one or more therapies. In specific embodiments, the terms “treat”,“treatment” and “treating” refer to the amelioration of at least onemeasurable physical parameter of a proliferative disorder, such asgrowth of a tumor, not necessarily discernible by the patient. In otherembodiments the terms “treat”, “treatment” and “treating” refer to theinhibition of the progression of a proliferative disorder, eitherphysically by, e.g., stabilization of a discernible symptom,physiologically by, e.g., stabilization of a physical parameter, orboth. In other embodiments the terms “treat”, “treatment” and “treating”refer to the reduction or stabilization of tumor size or cancerous cellcount.

Within the meaning of the present disclosure, the term “treat” alsodenotes to arrest, delay the onset (i.e., the period prior to clinicalmanifestation of a disease) and/or reduce the risk of developing orworsening a disease. The term “protect” is used herein to mean prevent,delay, or treat, or all, as appropriate, development, continuance oraggravation of a disease in a subject, e.g., a mammal or human.

The term “subject” or “patient” as used herein is intended to includeanimals, which are capable of suffering from or afflicted with a canceror any disorder involving, directly or indirectly, a cancer. Examples ofsubjects include mammals, e.g., humans, apes, monkeys, dogs, cows,horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenicnon-human animals. In a preferred embodiment, the subject is a human,e.g., a human suffering from, at risk of suffering from, or potentiallycapable of suffering from a proliferative disease, such as cancer.

The term “inhibition”, “inhibitor,” or “antagonist” includes a reductionin a certain parameter, e.g., an activity, of a given molecule orpathway. For example, inhibition of an activity of a targeted kinase(Raf or MEK) by 5%, 10%, 20%, 30%, 40% or more is included by this term.Thus, inhibition may be, but need not be, 100%.

As used herein, “salts” (which, what is meant by “or salts thereof” or“or a salt thereof”), can be present alone or in mixture with freecompounds of the combination of the invention, e.g., Raf inhibitorCompound with formula (I) or Raf inhibitor Compound with formula (II) orMEK inhibitor, preferably trametinib, and are preferablypharmaceutically acceptable salts. Such salts are formed, for example,as acid addition salts, preferably with organic or inorganic acids, fromcompounds of the combination of the invention with a basic nitrogenatom, especially the pharmaceutically acceptable salts. The term“pharmaceutically acceptable salts” refers to salts that retain thebiological effectiveness and properties of the compound and whichtypically are not biologically or otherwise undesirable. The compoundmay be capable of forming acid addition salts by virtue of the presenceof an amino group.

Lists of suitable salts can be found, e.g., in “Remington'sPharmaceutical Sciences”, 20th ed., Mack Publishing Company, Easton,Pa., (1985); and in “Handbook of Pharmaceutical Salts: Properties,Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany,2002).

For isolation or purification purposes it is also possible to usepharmaceutically unacceptable salts, for example picrates orperchlorates. For therapeutic use, only pharmaceutically acceptablesalts or free compounds are employed (where applicable in the form ofpharmaceutical preparations), and these are therefore preferred. In viewof the close relationship between the novel compounds in free form andthose in the form of their salts, including those salts that can be usedas intermediates, for example in the purification or identification ofthe novel compounds, any reference to the free compounds is to beunderstood as referring also to the corresponding salts, as appropriateand expedient. The salts of compounds used in the combination of theinvention are preferably pharmaceutically acceptable salts; suitablecounter-ions forming pharmaceutically acceptable salts are known in thefield. Unless otherwise specified, or clearly indicated by the text,reference to therapeutic agents useful in the pharmaceutical combinationprovided herein includes both the free base of the compounds, and allpharmaceutically acceptable salts of the compounds.

As used herein, the term “solvate” refers to a complex of variablestoichiometry formed by a solute (in this invention, trametinib) or asalt thereof and a solvent. Such solvents for the purpose of theinvention may not interfere with the biological activity of the solute.Examples of suitable solvents include, but are not limited to, water,methanol, dimethylsulforide, ethanol and acetic acid. Examples ofsuitable pharmaceutically acceptable solvents include, withoutlimitation, water, ethanol and acetic acid.

The term “synergistic effect” as used herein, refers to action of twoagents such as, for example, Raf inhibitor Compound with formula (I), ora pharmaceutically acceptable salt thereof, and a MEK inhibitor,preferably trametinib, or a pharmaceutically acceptable salt thereof, orRaf inhibitor Compound with formula (II), or a pharmaceuticallyacceptable salt thereof, and a MEK inhibitor, preferably trametinib, ora pharmaceutically acceptable salt thereof, to produce an effect, forexample, slowing the symptomatic progression of cancer or symptomsthereof, which is greater than the simple addition of the effects ofeach drug administered by themselves.

In one embodiment, the combination of the invention comprises (a) a Rafinhibitor compound selected from the group consisting of (i) Compound offormula (I)

or a pharmaceutically acceptable salt thereof, and

(ii) Compound of formula (II)

or a pharmaceutically acceptable salt thereof and (b) a MEK inhibitor.

In one embodiment, the combination of the invention comprises (a) a Rafinhibitor Compound of formula (I)

or a pharmaceutically acceptable salt thereof, and (b) a MEK inhibitor.

In one embodiment, the combination of the invention comprises (a) a Rafinhibitor Compound of formula (II)

or a pharmaceutically acceptable salt thereof, and (b) a MEK inhibitor.

In one embodiment, the combination of the invention comprises (a) a Rafinhibitor Compound of formula (I)

or a pharmaceutically acceptable salt thereof, and (b) a MEK inhibitorselected from the group comprising trametinib, PD0325901, PD184352,Refametinib, Cobimetinib, AS-701255, AS-701173, Pimasertib, RDEA436,R04987655, RG 7167 and RG7420, or a pharmaceutically acceptable salt orsolvate thereof.

In one embodiment, the combination of the invention comprises (a) a Rafinhibitor Compound of formula (II)

or a pharmaceutically acceptable salt thereof, and (b) a MEK inhibitorselected from the group comprising trametinib, PD0325901, PD184352,Refametinib, Cobimetinib, AS-701255,AS-701173, Pimasertib, RDEA436,R04987655, RG 7167 and RG7420, or a pharmaceutically acceptable salt orsolvate thereof.

In a preferred embodiment, the combination of the invention comprises(a) a Raf inhibitor compound selected from the group consisting of (i)Compound of formula (I)

or a pharmaceutically acceptable salt thereof, and (ii) Compound offormula (II)

or a pharmaceutically acceptable salt thereof and (b) MEK inhibitortrametinib, or a pharmaceutically acceptable salt or solvate thereof.

In a very preferred embodiment, the combination of the inventioncomprises (a) a Raf inhibitor Compound of formula (I)

or a pharmaceutically acceptable salt thereof, and (b) MEK inhibitortrametinib, or a pharmaceutically acceptable salt or solvate (e.g. thedimethyl sulfoxide solvate) thereof.

In another preferred embodiment, the combination of the inventioncomprises (a) a Raf inhibitor Compound of formula (II)

or a pharmaceutically acceptable salt thereof, and (b) MEK inhibitortrametinib, or a pharmaceutically acceptable salt or solvate thereof.

Combinations of the invention demonstrated increased depth anddurability of tumor response compared to either single-agent therapy incell lines and human xenograft models, Calu-6 (see Examples), and maytherefore be effective for the treatment of proliferative disease,particularly a cancer. Accordingly, the invention provides compositionsand methods using a Raf inhibitor selected from the group consisting ofCompound of formula (I), or a pharmaceutically acceptable salt thereof,and Compound of formula (II), or a pharmaceutically acceptable saltthereof, in combination with a MEK inhibitor, and in particular withtrametinib, or a pharmaceutically acceptable salt or solvate thereof,for treating solid tumors, particularly tumors that harbor one or moreMAPK pathway alterations, e.g. BRAF-mutant, KRAS-mutant and NRAS-mutantcancers.

Preferably, these therapeutic agents are administered at therapeuticallyeffective dosages which, when combined, provide a beneficial effect. Thepresent invention particularly pertains to a combination of theinvention useful for separate, simultaneous or sequential administrationto a subject in need thereof for treating a proliferative disease.Alternatively stated, the present invention particularly pertains to acombination of the invention for use in the treatment of a proliferativedisease.

The nature of proliferative disease is multifactorial. Under certaincircumstances, therapeutic agents with different mechanisms of actionmay be combined. However, just considering any combination oftherapeutic agents having different mode of action does not necessarilylead to combinations with advantageous effects.

In the present invention, the administration of the combination of theinvention is expected to result in a more beneficial effect, e.g., asynergistic or improved anti-proliferative effect, e.g., with regard tothe delay of progression or inhibiting the proliferative disease or itssymptoms, and maybe also further beneficial effects, e.g., fewerside-effects, e.g., an improved quality of life or e.g., decreasedmorbidity, as compared to either monotherapy.

The therapeutic agents of the combination of the invention may beseparately, simultaneously or sequentially administered to a subject inneed thereof. Preferably, these therapeutic agents are administered attherapeutically effective dosages which, when combined, provide abeneficial effect. Thus, in one embodiment of the present invention, thecombination of the invention is for use in the treatment of aproliferative disease, particularly a cancer.

In one embodiment, the proliferative disease is a cancer. The term“cancer” is used herein to mean a broad spectrum of tumors, includingall solid and hematological malignancies. The cancer may be at an early,intermediate or late stage. The cancer may be locally advanced ormetastatic.

The cancer to be treated by the combination therapy described herein mayhave progressed following standard of care or for whom no effectivestandard therapy exists.

The cancer to be treated by the combinations described herein may nolonger respond to treatment with BRAF inhibitors such as vemurafenib,dabrafenib and/or MEK inhibitors such as cobimetinib and trametinib. Forexample, the cancer may be melanoma, e.g. BRAFV600-mutant (includingBRAFV600E-mutant) melanoma which is refractory to treatment with acombination of dabrafenib and trametinib or which is refractory totreatment with a combination of cobimetinb and vemurafenib. The NSCLC,e.g. BRAFV600-mutant (including BRAFV600E-mutant) NSCLC, to be treatedby the combinations described herein may be refractory to treatment witha combination of BRAF inhibitors such as dabrafenib and MEK inhibitorssuch as trametinib.

In one embodiment the cancer is selected from the group comprisingmelanoma, non-small-cell lung cancer (NSCLC), colorectal cancer (CRC)including MUTYH-associated polyposis (MAP), pancreatic ductaladenocarcinoma (PADC), cervical cancer and ovarian cancer.

In one embodiment, the proliferative disease is non-small cell lungcancer (NSCLC).

In one embodiment, the proliferative disease is melanoma.

In one embodiment, the proliferative disease is colorectal cancer (CRC),including MUTYH-associated polyposis (MAP).

In one embodiment, the proliferative disease is pancreatic ductaladenocarcinoma (PADC).

In one embodiment, the proliferative disease is cervical cancer.

In one embodiment, the proliferative disease is ovarian cancer.

The combination of the invention is particularly useful for thetreatment of a proliferative disease such as a cancer that harbors oneor more Mitogen-activated protein kinase (MAPK) pathway alterations,such as KRAS-mutant tumors and NRAS-mutant tumors, and in particular,tumors expressing at least one gain-of-function mutation of Ras, asdescribed herein, and/or at least one gain-of-function mutation of Raf,as described herein.

Included are cancers or tumors having BRAF mutations, including V600D.V600E, V600K and others, e.g., NSCLC having at least one V600E or otherBRAF mutation, whether typical or atypical, i.e. BRAFV600E-mutant NSCLCor BRAF non-V600E-mutant NSCLC. Most of the BRAF mutations are clusteredto two regions: the glycine-rich P loop of the N lobe and the activationsegment and flanking regions.V600E mutation has been detected in avariety of cancers, and is due to a substitution of thymine with adenineat nucleotide 1799. This leads to valine (V) being substituted for byglutamate (E) at codon 600 (now referred to as V600E). BRAF-mutantmelanoma includes BRAFV600E-mutant and BRAFV600D-mutant melanoma.

Included are KRAS-mutant cancers or tumors. The term “KRAS-mutant” tumoror cancer includes any tumor that exhibits a mutated KRAS protein, inparticular gain of function KRAS-mutation; especially any G12X, G13X,Q61X or A146X KRAS-mutant, where X is any amino acid other than the onenaturally occurring at that position. E.g., a G12V mutation means that aGlycine is substituted with Valine at codon 12. Examples of KRASmutations in tumors include Q61H, Q61K, G12V, G12C, G12D, G12R, G12S,G13D, and A146T. Thus KRAS-mutant NSCLC includes tumors having at leastone KRAS mutation corresponding to G12X, G13X, Q61X or A146X,particularly at least one KRAS mutation selected from Q61K, G12V, G12Cand A146T NSCLC. The cancer may be at an early, intermediate or latestage.

KRAS-mutant cancers include KRAS G12D-mutant ovarian cancer; KRASG12V-mutant or G13D-mutant colorectal cancer; KRAS Q61H-mutant, KRASQ61K-mutant, KRAS G12C-mutant, KRAS G12S-mutant or KRAS G12V-mutantNSCLC; KRAS G12D-mutant, G12V-mutant, or KRAS G12R-mutant pancreaticcancer.

Included are NRAS mutant cancers or tumors.The term “NRAS-mutant” tumoror cancer includes any tumor that exhibits a mutated NRAS protein, inparticular gain of function NRAS-mutation; especially any G13R, Q61K,Q61L, Q61R, NRAS-mutant tumor. Thus NRAS-mutant melanoma includesmelanoma having at least one NRAS mutation corresponding to Q61K, Q61Lor Q61R. The cancer may be NRAS QG13R-mutant melanoma. The cancer may beat an early, intermediate or late stage. The cancer may be locallyadvanced or metastatic.

In one embodiment of the invention, the cancer is characterized by oneor more mutation in B-Raf.

In another embodiment cancer is resistant or refractory to standard ofcare.

In another embodiment cancer is resistant or refractory to treatmentwith a B-Raf inhibitor, e.g. dabrafenib.

In another embodiment cancer is resistant or refractory to treatmentwith a MEK inhibitor, e.g. trametinib.

In another embodiment cancer is resistant or refractory to treatmentwith a B-Raf inhibitor, e.g. dabrafenib, and a MEK inhibitor, e.g.trametinib.

In one embodiment, the cancer is characterized by at least one mutationselected from the group comprising BRAF and KRAS proteins.

In one embodiment, the cancer is characterized by a mutation selectedfrom the group consisting of BRAF, NRAS, KRAS mutation and combinationsthereof.

In one embodiment, the combination of the invention relates to a methodfor treating a proliferative disease, particularly a cancer.

The combination of the invention may be especially useful in treatingKRAS-mutant NSCLC, KRAS-mutant pancreatic cancer, KRAS-mutant colorectalcancer, or NRAS-mutant melanoma. In a preferred embodiment, theproliferative disease or the cancer to be treated is KRAS-mutant NSCLC.In another preferred embodiment, the proliferative disease or the cancerto be treated is NRAS-mutant melanoma.

In one embodiment, provided herein is a method for treating cancer in asubject in need thereof comprising administering a therapeuticallyeffective amount of a pharmaceutical combination of the inventioncomprising (a) a Raf inhibitor selected from the group consisting of (i)Compound of formula (I), as defined herein, or a pharmaceuticallyacceptable salt thereof, and (ii) Compound of formula (II), as definedherein, or a pharmaceutically acceptable salt thereof, and (b) a MEKinhibitor. In a preferred embodiment, the MEK inhibitor is trametinib,or a pharmaceutically acceptable salt or solvate thereof.

In an embodiment, provided herein is a method for treating cancer in asubject in need thereof administering simultaneously, separately orsequentially to a subject in need thereof a combination of the inventionin a quantity which is jointly therapeutically effective against saidproliferative disease comprising (a) a Raf inhibitor selected from thegroup consisting of (i) Compound of formula (I), as defined herein, or apharmaceutically acceptable salt thereof and (ii) Compound of formula(II), as defined herein, or a pharmaceutically acceptable salt thereof,and (b) a MEK inhibitor. In a preferred embodiment, the MEK inhibitor istrametinib, or a pharmaceutically acceptable salt or solvate thereof.

In a further embodiment, the present invention is particularly relatedto a method of treating a cancer harboring one or more Mitogen-activatedprotein kinase (MAPK) pathway alterations. In one embodiment, thepresent invention is related to a method of treating a cancer, which ischaracterized by at least one mutation selected from the groupcomprising BRAS, NRAS and KRAS proteins. In one embodiment, the presentinvention relates to the use of the combination of the invention for thepreparation of a medicament for the treatment of a proliferativedisease, particularly a cancer. In one embodiment, the combination ofthe invention is for use in the preparation of a medicament for thetreatment of cancer.

In a further embodiment, the present invention relates to the use of thecombination of the invention for the preparation of a medicament for thetreatment of a cancer characterized by gain-of-function mutation in theMAPK pathway.

In an embodiment, the combination or composition, or both, providedherein display a synergistic effect.

Accordingly, in one aspect, the invention may provide a method ofenhancing the efficacy of an anticancer compound by using it incombination with another anticancer compound, particularly a methodusing a Raf inhibitor selected from the group consisting of (i) Compoundof formula (I), as defined herein, or a pharmaceutically acceptable saltthereof, and (ii) Compound of formula (II), as defined herein, or apharmaceutically acceptable salt thereof, together with a MEK inhibitor,suitably trametinib, or a pharmaceutically acceptable salt or solvatethereof, to provide enhanced efficacy not safely achievable byadministering similar doses of either Compound of formula (I), or apharmaceutically acceptable salt thereof, or Compound (II), or apharmaceutically acceptable salt thereof, or the MEK inhibitor as asingle agent (monotherapy).

A further benefit may be that lower doses of the therapeutic agents ofthe combination of the invention can be used, for example, such that thedosages may not only often be smaller, but also may be applied lessfrequently, or can be used in order to diminish the incidence ofside-effects observed with one of the combination partners alone. Thisis in accordance with the desires and requirements of the patients to betreated.

In some embodiments, the Raf inhibitor compound selected from the groupconsisting of (i) Compound of formula (I), as defined herein, or apharmaceutically acceptable salt thereof, and (ii) Compound of formula(II), as defined herein, or a pharmaceutically acceptable salt thereof,or a pharmaceutically acceptable salt thereof, and/or the MEK inhibitor,preferably trametinib, or a pharmaceutically acceptable salt or solvatethereof, may be administered at a therapeutic or lower-than therapeuticdose relative to a single-agent dose level. In certain embodiments, theconcentration or dosage of the one therapeutic agent that is required toachieve inhibition, e.g., growth inhibition or tumor shrinkage is lowerwhen the other therapeutic agent is used or administered in combinationwith the first therapeutic agent than when each therapeutic agent isadministered individually. In certain embodiments, in a combinationtherapy, the concentration or dosage of one therapeutic agent that isrequired to achieve inhibition, e.g., growth inhibition, is lower thanthe therapeutic dose as a monotherapy, e.g., 10-20%, 20-30%, 30-40%,40-50%, 50-60%, 60-70%, 70-80%, or 80-90% lower.

In determining a synergistic interaction between one or more components,the optimum range for the effect and absolute dose ranges of eachcomponent for the effect may be definitively measured by administrationof the components over different w/w ratio ranges and doses to patientsin need of treatment. For humans, the complexity and cost of carryingout clinical studies on patients may render the use of this form oftesting as a primary model for synergy impractical. However, theobservation of synergy in certain experiments (see, e.g., Example 2 andExample 6) can be predictive of the effect in other species, and animalmodels exist may be used to further quantify a synergistic effect. Theobservation of synergy in one species can be predictive of the effect inother species and using animal models, as described herein, asynergistic effect can be measured and the results of such studies canalso be used to predict effective dose ratio ranges and the absolutedoses and plasma concentrations required in other species by theapplication of pharmacokinetic/pharmacodynamic (PK/PD) methods.Established correlations between tumor models and effects seen in mansuggest that synergy in animals may be demonstrated, for example, byxenograft models or in appropriate cell lines. It can be shown byestablished test models that a combination of the invention results inthe beneficial effects described herein. The person skilled in the artis fully enabled to select a relevant test model to prove suchbeneficial effects. The pharmacological activity of the combination ofthe invention may, for example, be demonstrated in a clinical study orin an in vivo or in vitro test procedure as essentially describedherein.

Administration of the combination includes administration of thecombination in a single formulation or unit dosage form, administrationof the individual agents of the combination concurrently but separately,or administration of the individual agents of the combinationsequentially by any suitable route. The individual combination partnersof the combination of the invention may be administered separately atdifferent times during the course of therapy, or sequentially in anyorder or concurrently in divided or single combination forms, e.g.,simultaneously or in jointly therapeutically effective amounts,preferably in synergistically effective amounts, e.g., in daily orintermittent (i.e., not daily) dosages corresponding to the amountsdescribed herein.

Compound of formula (I), or a pharmaceutically acceptable salt thereof,and Compound of formula (II), or a pharmaceutically acceptable saltthereof, for use in the methods, treatments, combinations andcompositions disclosed herein are potent inhibitors of BRAF and CRAF. Insome embodiments, Compound of formula (I), or a pharmaceuticallyacceptable salt thereof, or Compound of formula (II), or apharmaceutically acceptable salt thereof, is administered orally. In oneembodiment, Compound of formula (I), or a pharmaceutically acceptablesalt thereof, or Compound of formula (II), or a pharmaceuticallyacceptable salt thereof, is administered at a dose of about 50-1200 mg(e.g., per day). Compound of formula (I), or a pharmaceuticallyacceptable salt thereof, or Compound of formula (II), or apharmaceutically acceptable salt thereof, can be administered at a unitdosage of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about500 mg about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, about 1000mg, about 1050 mg, about 1100 mg, about 1150 mg or about 1200 mg. Theunit dosage of Compound of formula (I), or a pharmaceutically acceptablesalt thereof, or Compound of formula (II), or a pharmaceuticallyacceptable salt thereof, may be administered once daily, or twice daily,or three times daily, or four times daily, with the actual dosage andtiming of administration determined by criteria such as the patient'sage, weight, and gender; the extent and severity of the cancer to betreated; and the judgment of a treating physician. Preferably, the unitdosage of Compound of formula (I) or Compound of formula (II) isadministered once daily. In another preferred embodiment, the unitdosage of Compound of formula (I) or Compound of formula (II) isadministered twice daily.

The MEK inhibitor as part of the combination according to the presentinvention will be administered to a subject in need thereof in atherapeutically effective amount.

In a preferred embodiment, the MEK inhibitor trametinib, or apharmaceutically acceptable salt or solvate thereof, administered aspart of the combination according to the present invention in a subjectin need thereof will be an amount selected from about 0.125 mg to about10 mg per day; suitably, the amount will be selected from about 0.25 mgto about 9 mg per day; suitably, the amount will be selected from about0.25 mg to about 8 mg; suitably, the amount will be selected from about0.5 mg to about 8 mg per day; suitably, the amount will be selected fromabout 0.5 mg to about 7 mg per day; suitably, the amount will beselected from about 1 mg to about 5 mg per day; suitably, the amountwill be about 1 mg or 2 mg per day. In a preferred embodiment,trametinib, trametinib, or a pharmaceutically acceptable salt or solvatethereof, is administered at daily dose of 0.5 mg, 1 mg or 2 mg per day.

Where doses or dosages are mentioned herein, the amount referred torefers to the amount of the therapeutic agent. For example, when a 2 mgdosage of trametinib is administered, and trametinib is administered ina tablet containing trametinib dimethyl sulfoxide, the tablet willcontain trametinib dimethyl sulfoxide equivalent to 2 mg trametinib.

In some embodiments, trametinib, or a pharmaceutically acceptable saltor solvate thereof, is administered orally. In one embodiment,trametinib is prepared for administration via oral delivery, and may beused in solvated form in dimethyl sulfoxide. In some embodiments, thecompound is prepared in tablet form for oral administration. The tabletscan be produced in a variety of dosages for flexible administration.

The unit dosage of trametinib, or a pharmaceutically acceptable salt orsolvate thereof, may be administered once daily, or twice daily, orthree times daily, or four times daily. The total daily dose oftrametinib, or a pharmaceutically acceptable salt or solvate thereof,e.g., the dimethyl sulfoxide solvate, may be administered once or twicea day.

For example, as part of the combination therapy, Compound of formula(I), or a pharmaceutically acceptable salt thereof, may be administeredat a total daily dose of about 50 mg, about 100 mg, about 200 mg, about300 mg, about 400 mg, about 500 mg, about 600 mg, or about 800 mg andtrametinib, e.g. in the dimethyl sulfoxide solvate form, may beadministered in a total daily dose of about 1.0 or 2.0 mg. The dailydose of the Compound of formula (1) may be administered once or twiceper day. Hence, a dose of about 200 mg of Compound of formula (I) may beadministered twice per day and (total daily dose about 400 mg) and adose of about 1.0 mg or about 2.0 mg of trametinib may be administeredonce per day. Alternatively, a dose a dose of about 200 mg of Compoundof formula (I) may be administered twice per day and (total daily doseabout 400 mg) and a dose of about 1.0 mg or about 2.0 mg of trametinibmay be administered twice per day.

Compound of formula (I), or a pharmaceutically acceptable salt thereof,and a MEK inhibitor, preferably trametinib, or a pharmaceuticallyacceptable salt or solvate thereof, or Compound of formula (II), or apharmaceutically acceptable salt thereof, and a MEK inhibitor,preferably trametinib, or a pharmaceutically acceptable salt or solvatethereof, can be used together according to methods disclosed herein. Thetwo compounds can be administered together or separately, depending onthe intended dosage amount and frequency of administration, since it iscontemplated that the treatments of the invention may be continued for 2days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks,or more than 4 weeks as deemed appropriate to the treating physician,and further as guided using methods described herein to determine asuitable dosage and administration frequency. Frequency of dosage mayvary depending on the compound used and the particular condition to betreated. In general, the use of the minimum dosage that is sufficient toprovide effective therapy is preferred and may be determined by criteriasuch as the patient's age, weight, and gender; the extent and severityof the cancer to be treated; and the judgment of a treating physician.Patients may generally be monitored for therapeutic effectiveness usingassays suitable for the condition being treated, which will be familiarto those of ordinary skill in the art.

The optimum ratios, individual and combined dosages, and concentrationsof the combination partners of the combination of the invention, (i.e.,Compound of formula (I), or a pharmaceutically acceptable salt thereof,and a MEK inhibitor, suitably trametinib, or a pharmaceuticallyacceptable salt or solvate thereof, or Compound of formula (II), or apharmaceutically acceptable salt thereof, and a MEK inhibitor, suitablytrametinib, or a pharmaceutically acceptable salt or solvate thereof,that yield efficacy without toxicity are based on the kinetics of thetherapeutic agents' availability to target sites and a variety offactors, including, but not limited to, the degree of advancement of thedisease; the age, body weight, general health, gender and diet of theindividual; the time and route of administration; and other medicationsthe individual is taking. Optimal dosages may be established usingroutine testing and procedures that are well known in the art. Forexample, a single bolus may be administered, several divided doses maybe administered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.

The therapeutic agents of the combination of the invention may beadministered by any appropriate route. It will be appreciated that thepreferred route may vary with, for example, the condition of therecipient of the combination and the cancer to be treated. It will alsobe appreciated that each of the therapeutic agents may be administeredby the same or different routes and that the therapeutic agents, e.g.,Compound of formula (I), or a pharmaceutically acceptable salt thereof,and the MEK inhibitor, suitably trametinib, or a pharmaceuticallyacceptable salt or solvate thereof, or Compound of formula (II), or apharmaceutically acceptable salt thereof, and the MEK inhibitor,suitably trametinib, or a pharmaceutically acceptable salt or solvatethereof, may be compounded together in a pharmaceutical composition.

Compound of formula (I), or a pharmaceutically acceptable salt thereof,and the MEK inhibitor, suitably trametinib, or a pharmaceuticallyacceptable salt or solvate thereof, or Compound of formula (II), or apharmaceutically acceptable salt thereof, and the MEK inhibitor,suitably trametinib, or a pharmaceutically acceptable salt or solvatethereof, can be used together as disclosed herein. The two therapeuticagents of the combination of the invention can be administered together(simultaneously), sequentially or separately.

Furthermore, it does not matter if the compounds are administered in thesame dosage form, e.g., one compound may be administered topically andthe other compound may be administered orally. Suitably, boththerapeutic agents are administered orally.

Thus in one embodiment, one or more doses of Compound of formula (I), ora pharmaceutically acceptable salt thereof, or one or more doses ofCompound of formula (II), or a pharmaceutically acceptable salt thereof,are administered simultaneously, sequentially or separately with one ormore doses of a MEK inhibitor, suitably trametinib, or apharmaceutically acceptable salt or solvate thereof.

In one embodiment, multiple doses of Compound of formula (I), or apharmaceutically acceptable salt thereof, or multiple doses of Compoundof formula (II), or a pharmaceutically acceptable salt thereof, areadministered simultaneously, sequentially or separately with multipledoses of a MEK inhibitor, suitably trametinib, or a pharmaceuticallyacceptable salt or solvate thereof.

In one embodiment, multiple doses of Compound of formula (I), or apharmaceutically acceptable salt thereof, or multiple doses of Compoundof formula (II), or a pharmaceutically acceptable salt thereof, areadministered simultaneously, sequentially or separately with one dose ofa MEK inhibitor, suitably trametinib, or a pharmaceutically acceptablesalt or solvate thereof.

In one embodiment, one dose of Compound of formula (I), or apharmaceutically acceptable salt thereof, or one dose of Compound offormula (II), or a pharmaceutically acceptable salt thereof, isadministered simultaneously, sequentially or separately with multipledoses of a MEK inhibitor, suitably trametinib, or a pharmaceuticallyacceptable salt or solvate thereof.

In one embodiment one dose of Compound of formula (I), or apharmaceutically acceptable salt thereof, or one dose of Compound offormula (II), or a pharmaceutically acceptable salt thereof, isadministered simultaneously, sequentially or separately with one dose ofa MEK inhibitor, suitably trametinib, or a pharmaceutically acceptablesalt or solvate thereof.

In all the above embodiments Compound of formula (I), or apharmaceutically acceptable salt thereof, or Compound of formula (II),or a pharmaceutically acceptable salt thereof, may be administered firstor the MEK inhibitor, suitably trametinib, or a pharmaceuticallyacceptable salt or solvate thereof, may be administered first.

While it is possible that, for use in therapy, the Raf inhibitorselected from the group comprising (i) Compound of Formula (I), or apharmaceutically acceptable salt thereof, and (ii) Compound of Formula(II), or a pharmaceutically acceptable salt thereof, and/or the MEKinhibitor, suitably trametinib, or a pharmaceutically acceptable salt orsolvate thereof, may be administered as the raw chemical, it is possibleto present the active ingredient as a pharmaceutical composition.Accordingly, in one embodiment, provided herein is a pharmaceuticalcomposition comprising (a) a Raf inhibitor compound selected from thegroup consisting of Compound of formula (I), or a pharmaceuticallyacceptable salt thereof, and Compound of formula (II), or apharmaceutically acceptable salt thereof, and (b) a MEK inhibitor,suitably trametinib, or a pharmaceutically acceptable salt or solvatethereof. In an embodiment, the pharmaceutical composition furthercomprises one or more pharmaceutically acceptable diluents, excipientsor carriers. The carrier(s), diluent(s) or excipient(s) must beacceptable in the sense of being compatible with the other ingredientsof the formulation, capable of pharmaceutical formulation, and notdeleterious to the recipient thereof. Such elements of thepharmaceutical compositions utilized may be presented in separatepharmaceutical combinations or formulated together in one pharmaceuticalcomposition. The combinations disclosed herein can be administeredtogether in a single composition or administered separately in two ormore different compositions, e.g., compositions or dosage forms asdescribed and the components may be administered as the sameformulation, or as separate formulations, alone, e.g., as indicatedabove, or in combination with one or more pharmaceutically acceptablecarriers by any suitable route.

Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound (e.g., Compound offormula (I), or a pharmaceutically acceptable salt thereof, Compound offormula (II), or a pharmaceutically acceptable salt thereof, or the MEKinhibitor, suitably trametinib, or a pharmaceutically acceptable salt orsolvate thereof, calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The unit dosageform may also be a fixed combination.

The effective dosage of each of the combination partners may requiremore frequent administration of one of the therapeutic agent as comparedto the other therapeutic agent in the combination. Therefore, to permitappropriate dosing, packaged pharmaceutical products may contain one ormore dosage forms that contain the combination of compounds, and one ormore dosage forms that contain one of the therapeutic agents of thecombination of the invention, but not the other therapeutic agent of thecombination of the invention.

When the combination partners, which are employed in the combination ofthe invention, are applied in the form as marketed as single drug, theirdosage and mode of administration can be in accordance with theinformation provided on the package insert of the respective marketeddrug, if not mentioned otherwise. Therefore, to permit appropriatedosing, packaged pharmaceutical products can contain one or more dosageforms that contain the combination of agents, and one or more dosageforms that contain one of the therapeutic agents of the combination, butnot the other therapeutic agent of the combination.

Also within the scope of the invention is a combination kit comprising,as therapeutic agents, the combination of the invention forsimultaneous, separate or sequential administration as described herein,together with one or more other elements: instructions for use; otherreagents for use with the combination of the invention; devices or othermaterials for preparing the compound for administration, such as amixing container; pharmaceutically acceptable carriers; and devices orother materials for administration to a subject, such as a syringe.

By the term “combination kit” or “kit of parts” as used herein is meantthe pharmaceutical composition or compositions that are used accordingto the invention. When both compounds are administered simultaneously,the combination kit can contain Compound of formula (I), or apharmaceutically acceptable salt thereof, and the MEK inhibitor,suitably trametinib, or a pharmaceutically acceptable salt or solvatethereof, or Compound of formula (II), or a pharmaceutically acceptablesalt thereof, and the MEK inhibitor, suitably trametinib, or apharmaceutically acceptable salt or solvate thereof, in a singlepharmaceutical composition, such as a tablet, or in separatepharmaceutical compositions. When Compound of formula (I), or apharmaceutically acceptable salt thereof, and the MEK inhibitor,suitably trametinib, or a pharmaceutically acceptable salt or solvatethereof, or Compound of formula (II), or a pharmaceutically acceptablesalt thereof, and the MEK inhibitor, suitably trametinib, or apharmaceutically acceptable salt or solvate thereof, are notadministered simultaneously, the combination kit will contain Compoundof formula (I), or a pharmaceutically acceptable salt thereof, Compoundof formula (II), or a pharmaceutically acceptable salt thereof, and theMEK inhibitor, suitably trametinib, or a pharmaceutically acceptablesalt or solvate thereof, in separate pharmaceutical compositions eitherin a single package or in separate pharmaceutical compositions inseparate packages.

In one embodiment of the invention the kit of parts comprising thefollowing components: (a) a Raf inhibitor compound selected from thegroup consisting of (i) Compound of formula (I), or a pharmaceuticallyacceptable salt thereof, in association with pharmaceutically acceptableexcipients, diluents and/or carriers, and (ii) Compound of formula (II),or a pharmaceutically acceptable salt thereof, in association withpharmaceutically acceptable excipients, diluents and/or carriers; and(b) a MEK inhibitor, preferably trametinib, or a pharmaceuticallyacceptable salt or solvate thereof, in association with apharmaceutically acceptable excipients, diluents or carrier, wherein thecomponents are provided in a form which is suitable for sequential,separate and/or simultaneous administration. The combination kit canalso be provided with instructions, such as dosage and administrationinstructions. Such dosage and administration instructions can be of thekinds that are provided to a doctor, for example by a drug productlabel, or they can be of the kinds that are provided by a doctor, suchas instructions to a patient.

Other features, objects, and advantages of the invention will beapparent from the description and drawings, and from the claims.

The following Examples illustrate the invention described above; theyare not, however, intended to limit the scope of the invention in anyway. The beneficial effects of the pharmaceutical combination of thepresent invention can also be determined by other test models known assuch to the person skilled in the pertinent art.

EXAMPLES

The Examples below are set forth to aid in the understanding of theinvention but are not intended, and should not be construed, to limitits scope in any way.

Example 1 N-(3 -(2-(2-hy droxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isoni cotinami de

-   Compound of formula (I) is a morpholine-substituted biaryl compound    of the following structure

-   Compound of formula (I) is Example 1156 in published PCT application    WO2014/151616, the contents of which are incorporated by reference.

Example 1A

-   Compound of formula (I) is a type II inhibitor of both b-Raf and    c-Raf.

TABLE 1 Half maximal inhibitory concentration (IC-50) of Compound offormula (I) on b-Raf and c-Raf Compound b-Raf IC-50 (μM) c-Raf FL IC-50(μM) Compound of formula (I) 0.00073 0.00020

Example 1B

-   Compound of formula (I) exhibits activity on numerous human cancer    cell lines that express mutations in the MAPK pathway as shown in    the following Table. Activity is especially strong on cell lines    that harbor at least one mutation in BRAF or RAS.

TABLE 2 Effect of Compound of formula (I) on proliferation in a panel ofhuman cancer cell lines. Cell Line IC₅₀ [μM] Tumor Type BRAF RAS A3750.24 Melanoma V600E WT WM2664 0.45 Melanoma V600D WT IPC298 0.25Melanoma WT NRAS Q61L HeyA8 0.21 Ovarian G464E KRAS G12D HCT116 0.47Colorectal WT KRAS G13D Calu-6 1.5 NSCLC WT KRAS Q61K HuP-T4 0.65Pancreas WT KRAS G12V PSN1 0.68 Pancreas WT KRAS G12R TCC-PAN2 0.42Pancreas WT KRAS G12R NCI-H2073 18.2 NSCLC WT WT HCC827 >20 NSCLC WT WTPC3 >20 Prostate WT WT Different tumor cell lines were treated with dosetitrations of Compound of formula (I) for 72 h, and cell proliferationwas determined using the CellTiter-Glo ™ luminescent cell viabilityassay.

Example 1C

-   To investigate the activity of Compound of formula (I) in B-Raf V600    mutant melanoma cells refractory to B-Raf and/or MEK inhibitors, the    anti-proliferative activity of Compound of formula (I) in the    mechanistic models derived from the B-Raf V600 melanoma cell line    A375 expressing mutations of MEK1/2, NRAS, or a splice variant of    BRAF was evaluated. These mutations have been demonstrated in both    preclinical studies and clinical samples to confer B-Raf and/or MEK    inhibitor resistance. Growth inhibitory effects of Compound of    formula (I) in the parental A375 cell line and its derivatives    expressing the various mutant alleles, in comparison with efficacy    of the B-Raf inhibitor vemurafenib and the MEK inhibitor    selumetinib, are summarized below. The mutations conferred    resistance to both the B-Raf and MEK inhibitors, leading to greater    than 50-fold increases in IC₅₀ values. In contrast, the resistant    models were still sensitive to Compound of formula (I), with only a    2-3 fold increase in IC₅₀. These data support the use of Compound of    formula (I) in B-Raf V600 melanoma patients who have become    refractory to B-Raf and/or MEK inhibitors.

TABLE 3 Anti-proliferative effect of Compound of formula (I) inmechanistic A375 models resistant to BRAF and MEK inhibitors Compound offormula (I) Vemurafenib Selumetinib IC₅₀ IC₅₀ IC₅₀ Cell Line [μM] [μM][μM] A375 0.42 0.066 0.036 A375/BRAFp61- 0.72 8.51 >10 V600E A375/MEK1Q56P 1.15 9.62 5.35 A375/MEK1 C121S 1.14 8.7 2.33 A375/MEK1 E203K 1.055.58 1.81 A375/MEK2 Q60P 1.12 5.28 4.84 A375/NRAS Q61K 0.95 9.38 5.5A375 cell lines were engineered to inducibly express resistance modelsafter treatment with doxycycline. Cell were then treated with serialdilutions of Compound of formula (I), vemurafenib or selumetinib for 72hours to assess anti-proliferative activity. Cell proliferation wasdetermined using the CellTiter-Glo ™ luminescent cell viability assayand calculated as percent of DMSO control.

Example 1D

Compound of formula (I) was formulated for oral dosing into tabletscontaining about 50 mg of Compound of formula (I) according toprinciples well known in the art. A number of tablets sufficient toprovide a desired dosage were administered once daily to fastedsubjects. Subjects were treated at doses of 100 mg once per day, or 200mg once per day. Serial blood samples for pharmacokinetic (PK)assessments were collected up to 48 hours after the first dose ofCompound of formula (I) (Cycle 1 Day 1), and up to 24 hours aftermultiple doses (Cycle 1 Day 15). Preliminary available data are asfollows. Maximum plasma concentrations (Cmax) of 447 ng/ml and 889 ng/mlwere achieved within 4 hours after administration of a single 100 mgdose and single 200 mg dose, respectively. Mean plasma exposure over thedose interval of 24 hours (AUCtau) on day 1 of dosing was 5679 hr.ng/mland 10019 hr.ng/ml after the 100 mg and 200 mg doses of Compound offormula (I), respectively. The half-life is calculated to be around23-24 hours in patients. The once daily dosing of 100 mg resulted inslight accumulation of Compound of formula (I) in plasma, with anaccumulation ratio of 1.8. Based on these data, a dosing schedule ofonce per day was established.

Example 2 Anti-Tumor Activity of Compound of Formula (I) in KRAS-MutantNSCLC Models

In cell-based assays, Compound of formula (I) has demonstratedanti-proliferative activity in cell lines that contain a variety ofmutations that activate MAPK signaling. For instance, Compound offormula (I) inhibited the proliferation of the non-small cell lungcancer cell line Calu-6 (KRAS Q61K) and colorectal cell line HCT116(KRAS G13D) with IC50 values ranging from 0.2-1.2 μM.

Activity of Compound of formula (I) was tested in vivo in severalxenograft models. As shown in FIGS. 1-4, Compound of formula (I) showedsingle agent activity in KRAS mutant lung cancer models.

Calu6 model (KRAS(Q61K) mutant NSCLC): Female nude tumor bearing Calu6mice, n=8 per group were randomized into treatment groups when theaverage tumor volume was 324 mm3. Treatment with Compound of formula (I)was initiated on Day 15 post xenograft implant. Animals wereadministered an oral dose of either vehicle, Compound of formula (I) at15 mg/kg twice a day (bid), 30 mg/kg once a day (qd), 50 mg/kg bid, 100mg/kg bid, 200 mg/kg qd or 300 mg/kg every other day (q2d) for 13consecutive days at a dosing volume of 10 ml/kg of animal body weightduring course of treatment. Tumor volumes were collected at the time ofrandomization and twice weekly thereafter for the study duration usingdigital calipers (FIG. 1). Slight body weight loss was observed in the200 mg/kg qd (4% body weight loss) and 300 mg/kg q2d (9% body weightloss) treatment groups.

H358 model (KRAS(G12C) mutant NSCLC): SCID beige female tumor-bearingNCI-H358 mice, n=8 per group, were randomized into 3 groups 14 days posttumor cell inoculation with an average tumor volume range of 261 mm³.

Animals were administered an oral dose of either vehicle, Compound offormula (I) at 30 mg/kg or at 200 mg/kg daily for 14 consecutive days ata dosing volume of 10 ml/kg of animal body weight during course oftreatment. Tumor volumes were measured by digital caliper 3 times a weekand body weights of all animals were recorded through the course oftreatment (FIG. 2). Slight body weight loss was observed in the 30 mg/kg(4% body weight loss) and 200 mg/kg (6% body weight loss) treatmentgroups.

PTX model HLUX1156-KRAS(G12C) mutant NSCLC: Nude female mice tumorbearing patient derived primary lung cancer xenograft HLUX1156, n=6 pergroup, were randomized into 2 groups with an average tumor volume rangeof 262 mm3. Treatment was initiated on Day 38 post xenograft implant.Animals were administered an oral dose of either vehicle or Compound offormula (I) at 100 mg/kg daily for 14 consecutive days at a dosingvolume of 10 ml/kg of animal body weight during course of treatment.Tumor volumes were measured by digital caliper 2 times a week and bodyweights of all animals were recorded through the course of treatment(FIG. 3). Treatment was well tolerated as judged by lack of significantbody weight loss.

H727 model-KRAS(G12V) mutant NSCLC: Foxn1 nude female mice tumor bearingNCI-H727, n=8 per group, were randomized into 2 groups with an averagetumor volume range of 275 mm3. Treatment was initiated on Day 21 postxenograft implant. Animals were administered an oral dose of eithervehicle or Compound of formula (I) at 100 mg/kg daily for 14 consecutivedays at a dosing volume of 10 ml/kg of animal body weight during courseof treatment. Tumor volumes were measured by digital caliper 3 times aweek and body weights of all animals were recorded through the course oftreatment (FIG. 4). Slight body weight loss was observed in the 100mg/kg (4.5% body weight loss) treatment groups.

Anti-tumor activity was determined by assessing percentage of tumorvolume in the treatment groups versus that in vehicle-treated (% T/C) orpercentage of tumor regression compared to the starting volume (%regression). In vivo, treatment with Compound of formula (I) resulted intumor regression in several human KRAS-mutant models including theNSCLC-derived Calu-6 (KRAS Q61K) and NCI-H358 (KRAS G12C) xenografts. Inall cases, anti-tumor effects were dose-dependent. The Calu-6 model wassensitive to Compound of formula (I) with tumor regression observed atdoses of 50 mg/kg and 100 mg/kg twice daily (BID) and 100 and 200 mg/kgonce daily (QD) and 300 mg/kg once every other day (Q2D) in mice.Regression was also achieved in a second human NSCLC model, NCI-H358, atthe 200 mg/kg QD dose. Furthermore, data from a dose fractionationefficacy study in Calu-6 xenografts demonstrated that across differentdosing levels, Compound of formula (I) dosed QD and fractioned twice aday (BID) showed similar levels of anti-tumor activity. These resultssupport exploration of QD or BID dose regimen in the clinic.

Collectively the in vitro and in vivo MAPK-pathway suppression andanti-proliferative activity observed for Compound of formula (I) atwell-tolerated doses suggests that Compound of formula (I) may haveanti-tumor activity in patients with tumors harboring activating lesionsin the MAPK pathway and in particular may therefore be useful as asingle agent or in combination with a second agent, such as an inhibitoraffecting a different step of the MAPK pathway, for the treatment ofNSCLC patients harboring KRAS mutations. Compound of formula (I) hasbeen shown to have activity as a single agent against various othercancers that express gain-of- function mutations in the MAPK pathway,e.g., in RAS or RAF, including ovarian cancer, pancreatic cancer, andmelanoma.

Example 2 Synergistic Effects of a Combination of Compound of Formula(I) and Trametinib on Cell Growth

The effects of combining the Compound of formula (I) and the MEK1/2inhibitor trametinib on the proliferation and signaling in NRAS mutantmelanoma, KRAS mutant NSCLC, KRAS mutant PDAC and KRAS mutant CRC weretested as follows.

The CellTiter-Glo® (CTG) Luminescent Cell Viability Assay kit (Promega,Madison, Wis., USA) measures the amount of ATP present in a well afterlysis of the cells. The ATP released upon lysis is measured in anenzymatic reaction which includes Luciferase and its substrateLuciferin. The amount of light emitted is proportional to the amount ofATP, which in turn is proportional to the number of live cells in thewell. This assay is used to determine the proportion of viable cellsafter drug treatment.

NRAS mutant melanoma cell lines and KRAS mutant NSCLC cell lines weremaintained in appropriate media.

Cell Line Mutation Media* Cancer Type Calu-6 KRAS^(Q61K) EMEM + 10% FBS NSCLC A549 KRAS^(G12S)   F12 + 10% FBS NSCLC HCC-2108 KRAS^(Q61H) RPMI +10% FBS NSCLC NCI-H2122 KRAS^(G12C) RPMI + 10% FBS NSCLC NCI-H23KRAS^(G12C) RPMI + 10% FBS NSCLC NCI-H358 KRAS^(G12C) RPMI + 10% FBSNSCLC NCI-H2030 KRAS^(G12C) RPMI + 10% FBS NSCLC Hs 944.T NRAS^(Q61K)RPMI + 10% FBS Melanoma IPC-298 NRAS^(Q61L) RPMI + 10% FBS MelanomaMEL-JUSO NRAS^(Q61L) RPMI + 10% FBS Melanoma MM127 NRAS^(G13R) RPMI +10% FBS Melanoma MM415 NRAS^(Q61L) RPMI + 10% FBS Melanoma MM485NRAS^(Q61R) RPMI + 10% FBS Melanoma SK-MEL-2 NRAS^(Q61R) EMEM + 10% FBS Melanoma *RPMI: Roswell Park Memorial Institute medium. FBS: FetalBovine Saline EMEM: Eagle's minimum essential mediumFor NRAS mutant melanoma lines, combinations were assessed in a fullgrid matrix using Compound of formula (I) concentrations ranging from0.002 to 10 μM and trametinib concentrations ranging from 1.52E-4 to 1μM. For KRAS mutant NSCLC lines, combinations were assessed in a fullgrid or in a checkerboard formatted matrix using Compound of formula (I)concentrations ranging from 0.014 to 10 μM and trametinib concentrationsranging from 0.004 to 2.7 μM. Whether or not the combination wassynergistic in a particular cell line was determined using synergy score(SS) and combination index (CI) at 50 percent inhibition (CI₅₀) effectsizes (Lehar et al., 2009). A summary of these values for each cell lineis shown in the Table below.

TABLE Summary of Compound of formula (I) × trametinib Synergy Scores andCI₅₀ values Loewe Loewe Synergy Cancer KRAS Synergy Score CI₅₀ SynergyCell Line Type Mutation Score Error CI₅₀ Error Determination Hs 944.TMelanoma NRAS^(Q61K) 3.48 0.13 0.34 0.06 Synergy IPC-298 MelanomaNRAS^(Q61L) 4.22 0.20 0.40 0.16 Synergy MEL-JUSO Melanoma NRAS^(Q61L)5.44 0.18 0.31 0.05 Synergy MM127 Melanoma NRAS^(G13R) 6.28 0.33 0.300.04 Synergy MM415 Melanoma NRAS^(Q61L) 2.69 0.11 0.29 0.04 SynergyMM485 Melanoma NRAS^(Q61R) 3.85 0.12 0.38 0.17 Synergy SK-MEL-2 MelanomaNRAS^(Q61R) 6.58 0.11 0.11 0.03 Synergy SK-MEL-30 Melanoma NRAS^(Q61K)4.18 0.12 0.00 0.00 Synergy A549 NSCLC KRAS^(G12S) 5.65 0.26 0.42 0.06Synergy Calu-6 NSCLC KRAS^(Q61K) 6.12 0.15 0.16 0.02 Synergy HCC-2108NSCLC KRAS^(Q61H) 4.16 0.12 0.64 0.05 Synergy NCI-H2122 NSCLCKRAS^(G12C) 6.67 0.18 0.34 0.02 Synergy NCI-H23 NSCLC KRAS^(G12C) 2.590.12 0.41 0.11 Synergy NCI-H358 NSCLC KRAS^(G12C) 2.17 0.06 0.28 0.04Synergy NCI- NSCLC KRAS^(G12C) 0.83 0.06 0.62 0.03 No synergy/additivity ^(@) Cell line likely not MAPK dependent as there was noeffect on single agent treatment either.The studies were repeated using KRAS-mutant PDAC and CRC models and thefollowing results obtained.

TABLE Summary of Compound of Formula I × trametinib Synergy Scores andCI₅₀ values in KRAS^(mut) PDAC and CRC Loewe Loewe Cancer KRAS SynergySynergy Synergy Cell Line Type Mutation Score Score Error CI₅₀ CI₅₀Error Determination HCC-56 CRC KRAS^(G12V) 3.12 0.16 0.88 0.07 SynergyHCT 116 CRC KRAS^(G13D) 5.57 0.15 0.39 0.03 Synergy HCT-15 CRCKRAS^(G13D) 6.27 0.34 0.16 0.05 Synergy SK-CO-1 CRC KRAS^(G12V) 2.340.13 0.36 0.04 Synergy CFPAC-1 PDAC KRAS^(G12V) 3.65 0.23 0.09 0.02Synergy HPAF-II PDAC KRAS^(G12D) 4.94 0.18 0.44 0.05 Synergy HUP-T4 PDACKRAS^(G12V) 4.21 0.15 0.00 0.00 Synergy MIA PaCa-2 PDAC KRAS^(G12C) 3.650.06 0.46 0.02 Synergy PSN1 PDAC KRAS^(G12R) 7.51 0.07 0.27 0.01 SynergyQGP1 PDAC KRAS^(G12V) 1.55 0.07 0.42 0.03 Additive/ Synergy SU.86.86PDAC KRAS^(G12D) 3.44 0.21 0.55 0.05 Synergy TCC-PAN2 PDAC KRAS^(G12R)3.34 0.12 0.00 0.00 SynergyA general guideline for interpretation of the scores/values is providedin the Table below.

Combination parameters Effect description SS > 3.0 and Best C.I. > 0.5Synergy SS > 2.0 and Best C.I. < 0.5 Synergy SS > 2.0 and Best C.I. >0.5 Additive/Synergy SS > 1.0 but < 2.0 and Best C.I. < 0.5Additive/Synergy SS < 1.0 and Best C.I. < 0.5 Additive

-   As demonstrated from the Tables above, the Compound of formula (I)    and trametinib have synergistic effects on cell growth in KRAS and    NRAS mutant cell lines. Combining Compound of formula (I) and    trametinib was moderately to strongly synergistic in all but one    cell lines tested. In the one NSCLC cell line, NCI-H2030, in which    no-synergy/additivity was observed, the lack of single agent    response to either Compound of formula (I) or trametinib is very    likely due to the fact that this model is not MAPK dependent.-   Combining Compound of formula (I) and trametinib was moderately to    strongly synergistic in all NRAS mutant melanoma cell. KRAS mutant    PDAC, KRAS mutant CRC lines tested. Combining Compound of    formula (I) and trametinib was moderately to strongly synergistic in    14 of 15 KRAS mutant NSCLC cell lines tested.-   In the example of NCI-H2122, which showed the strongest synergy of    the KRAS mutant NSCLC lines tested, the Loewe excess grid displays a    difference of >40% between the Loewe dose additivity model and    observed values at low doses of Compound of Formula (I) and    Trametinib (0.041 to 0.37 μM Compound of Formula (I) and 0.11 μM    Trametinib). In fact, single agent Compound of formula (I) has    little to no activity at these low doses, with maximum inhibition    values <30% below the 1 μM dose but the combination with trametinib    at the same Compound of formula (I) doses has significant synergy    (with Loewe excess >40%).-   In SK-MEL-2, which had the strongest synergy of all NRAS melanoma    cell lines tested, there is significant synergy at low doses of both    trametinib and Compound of formula (I) (0.002 to 0.371 μM Compound    of Formula (I) and 0.000152 to 0.001 μM of Trametinib). At the two    lowest doses of trametinib (0.000152 - 0.000457 μM of Trametinib),    there is strong synergy with Compound of formula (I) doses ranging    from 0.014 to 0.124 μM, leading to anti-proliferative effects at    concentrations where there is little to no single agent activity    observed.    Compound of Formula (I) and Trametinib Inhibit Pharmacodynamic    Biomarkers in KRAS and NRAS Mutant Cell Lines-   To probe the mechanism underlying the synergistic anti    -proliferative effects of combining Compound of formula (I) and    trametinib, the effects of this combination on MAPK signaling was    investigated via western blot analysis. Cells were treated with    single agent and combination doses of Compound of formula (I) (300    nM) and trametinib (3 nM) for 4 or 24 hours. As a further comparison    to the combination doses, cells were also treated with 10-fold    higher single agent doses of 3000 nM and 30 nM for Compound of    formula (I) and trametinib, respectively.-   In both NRAS mutant melanoma (IPC-298 and MM415) and KRAS mutant    NSCLC (NCI-H23 and NCI-H358) cell lines examined, treatment of cells    with the combination of Compound of formula (I) and trametinib was    superior to either single agent alone at suppressing the pathway, as    judged by the stronger suppression of pMEK1/2 and pERK1/2 levels in    the combination compared to either single agent treatment. Moreover,    suppression generated by the low dose combination was superior to    what could be achieved at 10-fold higher levels of Compound of    formula (I), and similar, albeit slightly less robust than was    observed for at the higher trametinib levels. Thus, combined    treatment of Compound of formula (I) and trametinib resulted in a    highly synergistic suppression of MAPK signaling.

Example 3 In vivo Anti-Tumor Activity of Combination of Compound ofFormula (I) with Trametinib.

As described below, the combined treatment with Compound of formula (I)and trametinib was found to lead to increased depth and durability oftumor response compared to either single agent in human KRAS mutantNSCLC, CRC, PDAC and NRAS mutant melanoma xenograft models. Therefore,the combination activity of Compound of Formula (I) and trametinib willlikely achieve greater and more durable responses in patients whosecancers harbor an activated MAPK pathway.

Anti-Tumor Activitiy of a Combination of Compound of Formula (I) and aMEK Inhibitor in KRAS-Mutant NSCLC Cancer Models

Anti-tumor activitiy of a combination of Compound of Formula (I) and aMEK inhibitor in KRAS-mutant NSCLC models Calu-6 NSCLC tumors wereestablished in nude female mice. When tumors reached approximately 250mm³, mice were randomized according to tumor volume into treatmentgroups (n=7). Treatment was initiated on Day 10 post xenograft implant.Anti-tumor activity was determined on day 27 post tumor cellimplantation; 17 days post initiation of treatment.

Test agents were administered orally once daily (qd) or once every otherday (q2d) at the dose levels indicated in FIGS. 5 and 6 at a dosingvolume of 10 ml/kg of animal body weight during course of treatment.Single agent was administered for 28 days, and the combination ofCompound of formula (I) and trametinib was administered for 56 days.Tumor volumes were measured by digital caliper 2 times a week and bodyweights of all animals were recorded throughout the course of treatment.

Anti-tumor activity, mean change in tumor volume, mean percent change inbody weight and survival 17 days post treatment initiation (27 days postimplant) are reported in the Table below.

TABLE Anti-tumor efficacy and tolerability of Compound of formula (I)and trametinib in the Calu-6 human NSCLC subcutaneous tumor xenograftmodel in mice on day 27 post-implantation Dose (mg/kg) % Change in tumorChange in body Survival and % Tumor volume (mm3) weight (%) (Survivors/Drug schedule T/C Regression Mean +/− SEM Mean +/− SEM total mice)Vehicle — 1086.91 ± 112.22  6.19 ± 0.93 7/7 Compound of 30 qd  26*282.92 ± 91.55 −2.26 ± 0.89 7/7 formula ((I) Trametinib 0.3 qd 36 395.32± 36.43 −1.55 ± 1.04 7/7 Trametinib 0.3 q2d 53 574.17 ± 30.67  1.71 ±1.01 7/7 Compound of 30 qd + 0.3 qd 41** −107.68 ± 21.01  −2.49 ± 1.607/7 formula (I) + Trametinib Compound of  30 qd + 0.3 q2d 13** −33.86 ±10.09 −3.43 ± 0.93 7/7 formula (I) + Trametinib Vehicle: Untreated qd:once aday q2d: every other day The experiment was evaluated on study day27. *p = 0.0039 (Compound of formula (I) 30 mg/kg qd versus Vehicletreated group), p < 0.0001 (Compound of formula (I) 30 mg/kg qd +trametinib 0.3 mg/kg qd, and Compound of formula (I) 30 mg/kg qd +trametinib 0.3 mg/kg q2d versus Vehicle treated group) (One-Way ANOVADunn's multiple comparison test).

Compound of formula (I) dosed at 30 mg/kg qd achieved 26% T/C, whiletrametinib dosed at either 0.3 mg/kg qd or 0.3 mg/kg q2d achieved 36%T/C and 53% T/C, respectively, 17 days post dosing. Combining Compoundof formula (I) dosed at 30 mg/kg qd with trametinib dosed at either 0.3mg/kg qd or 0.3 mg/kg q2d achieved 41% and 13 regressions, respectively,17 days post dosing (FIG. 5).

To quantitatively assess the time to tumor progression amongst groups,the day that tumors surpassed an arbitrary cut-off of 700mm³ in volumewas noted. Again the combination of Compound of Formula (I) with eitherdose of trametinib achieved increased, statistically significantanti-tumor activity when compared to single agents. While tumors in micedosed with Compound of formula (I) and trametinib as single agentsprogressed under treatment, the combination of Compound of formula (I)and trametinib together maintained tumor regressions for longer thanmonotherapy- with significant anti-tumor activity 28 days post dosing(38 days post tumor implantation) when compared to single agents.

All treatment groups were also well tolerated with minimal body weightloss for the duration of the study. In addition to the increased depthof response, the combination of Compound of formula (I) and trametinibalso led to an increased durability of response (Table below and FIG.6).

TABLE Anti-tumor efficacy and tolerability of Compound of formula (I)and trametinib in the Calu-6 human NSCLC subcutaneous tumor xenograftmodel in mice on day 38 post-implantation % Change in tumor Change inbody Survival Day to median Dose (mg/kg) % Tumor volume (mm3) weight (%)(Survivors/ tumor volume Drug and schedule T/C Regression Mean +/− SEMMean +/− SEM total) ~700 mm³ Vehicle — — — — Compound of 30 qd 76 —1040.54 ± 194.73 2.72 ± 0.66 7/7 31 formula (I) trametinib 0.3 q2d 100 — 1364.31 ± 107.51 2.75 ± 1.93 7/7 24 trametinib 0.3 qd 70 — 961.52 ±39.00 2.51 ± 1.48 7/7 27 Compound of 30 qd + 0.3 q2d  14* — 195.78 ±28.15 2.57 ± 2.88 7/7 45 formula (I) + trametinib Compound of  30 qd +0.3 qd 17** −44.63 ± 17.46 2.59 ± 1.50 7/7 59 formula (I) + trametinibVehicle: Untreated qd: once a day q2d: every other day % T/C wascalculated using trametinib 0.3 mg/kg q2d group as a control Theexperiment was evaluated on study day 38. *p = 0.0041 (Compound offormula (I) 30 mg/kg qd + trametinib 0.3 mg/kg q2d versus trametinib 0.3mg/kg q2d treated group), **p < 0.0001 (Compound of formula (I) 30 mg/kgqd + trametinib 0.3 mg/kg qd versus trametinib 0.3 mg/kg q2d treatedgroup) (One-Way ANOVA Dunn's multiple comparison test).Anti-Tumor Activitiy of a Combination of Compound of Formula (I) and aMEK Inhibitor in KRAS-Mutant Colorectal Cancer Models

HCT116 (KRAS G13D) colorectal cancer (CRC) tumors were established innude female mice. When tumors reached approximately 230 mm³, mice wererandomized according to tumor volume into treatment groups (n=6).Treatment was initiated on Day 14 post xenograft implant. Anti-tumoractivity was determined on day 31 post tumor cell implantation (the lastday of vehicle treated mice).

Test agents were administered orally once daily (qd) or once every otherday (q2d) at the dose levels indicated in FIG. 16 (A) at a dosing volumeof 10 ml/kg of animal body weight during course of treatment. Tumorvolumes were measured by digital caliper 2 times a week and body weightsof all animals were recorded throughout the course of treatment.Compound of formula (I) dosed at 100 mg/kg qd achieved 30% T/C, whiletrametinib dosed at 0.3 mg/kg qd achieved 44% T/C, 17 days post dosing.Combining Compound of formula (I) dosed at 100 mg/kg qd with trametinibdosed at 0.3 mg/kg q2d achieved 26% regression, 17 days post dosing(FIG. 16). On day 24 post tumor implantation, one mouse was sacrificeddue to increased body weight loss. On day 31, the last day of vehicletreatment, mice treated with Compound of formula (I) had a combined bodyweight loss of 4%; mice treated with trametinib exhibited 3.5% bodyweight loss, and mice treated with the combination of Compound offormula (I) and trametinib showed 9% body weight loss .

Anti-Tumor Activitiy of a Combination of Compound of Formula (I) and aMEK Inhibitor in PDAC Cancer Models

Patient derived 2043 PDAC (KRAS^(G12D)) tumors were established in nudefemale mice. When tumors reached approximately 230 mm³, mice wererandomized according to tumor volume into treatment groups (n=6).Treatment was initiated on Day 52 post xenograft implant. Anti-tumoractivity was determined on day 73 post tumor cell implantation.

Test agents were administered orally once daily (qd) or once every otherday (q2d) at the dose levels indicated in FIG. 17 A at a dosing volumeof 10 ml/kg of animal body weight during course of treatment. Tumorvolumes were measured by digital caliper 2 times a week and body weightsof all animals were recorded throughout the course of treatment.Compound of formula (I) dosed at 100 mg/kg qd achieved 55% T/C, whiletrametinib dosed at 0.3 mg/kg qd achieved 32% T/C, 21 days post dosing.Combining Compound of formula (I) dosed at 100 mg/kg qd with trametinibdosed at 0.3 mg/kg q2d achieved 35 regression, 21 days post dosing (FIG.17). On day 66 post tumor implantation, one mouse was sacrificed due toincreased body weight loss. On day 73, the last day of combinationtreatment, mice treated with compound of formula I had a combined bodyweight loss of 0.6%; mice treated with tramentinib exhibited 0.3% bodyweight gain, and mice treated with the combination of compound offormula I and trametinib showed 9.6% body weight loss.

-   Anti-tumor activitiy of a combination of Compound of Formula (I) and    a MEK inhibitor in melanoma cancer models-   The anti-tumor activity of the combination of Compound of    Formula (I) and a MEK inhibitor in several human NRAS mutant    melanoma xenografts was studied as follows.

(i) SKMEL30 melanoma models

SKMEL30 (NRAS^(Q61K)) melanoma tumors were established in nude femalemice. Treatments were administered at a dose volume of 10 mL/kg. Micewere randomized into treatment groups (n=9) on day 12 following tumorimplantation, when the average tumor volume was 190 mm3. Anti-tumoractivity was determined on day 34 post tumor cell implantation; 22 dayspost initiation of treatment

Test agents were administered orally once (qd) or twice daily (bid) atthe dose levels indicated in FIG. 18 at a dosing volume of 10 ml/kg ofanimal body weight during course of treatment. Tumor volumes weremeasured by digital caliper 2 times a week and body weights of allanimals were recorded throughout the course of treatment. On day 34, thelast day that the vehicle treated group was on study, Compound offormula (I) treatment resulted in 5% tumor regression, while 0 .3 mg/kgqd of Trametinib resulted in8%T/C. The combination of Compound offormula (I) with trametinib at 0.15 mg/kg qd led to a further increasedanti-tumor activity of 48% tumor regression when compared to the vehicletreated group (FIG. 18). All treatment groups were well tolerated withminimal body weight loss for the duration of the study. Single agentgroups were dosed continuously for the duration of the study; in theCompound of formula I and Trametinib combination group, a brief dosingholiday of Trametinib only (day 28 till day 31) was provided after whichthe full combination was resumed to the end of the study

(ii) NRAS-mutant melanoma model 20667

Patient derived “20667” (NRAS^(Q61R)) melanoma tumors were establishedin nude female mice. When tumors reached approximately 300 mm³, micewere randomized according to tumor volume into treatment groups (n=7).Treatment was initiated on Day 17 post xenograft implant. Anti-tumoractivity of the single agents was determined on day 34 post tumorimplantation, while the anti-tumor activity of the combination betweenCompound of formula (I) and trametinib was determined on day 48 posttumor cell implantation.

Test agents were administered orally once daily (qd) or twice daily(bid) at the dose levels indicated in FIG. 19 at a dosing volume of 10ml/kg of animal body weight during course of treatment. Tumor volumeswere measured by digital caliper 2 times a week and body weights of allanimals were recorded throughout the course of treatment. Compound offormula (I) dosed at 100 mg/kg qd achieved 87% T/C, while trametinibdosed at 0.3 mg/kg qd achieved 82% T/C, 17 days post dosing. CombiningCompound of formula (I) dosed at 50 mg/kg bid with trametinib dosed at0.3 mg/kg qd achieved 68% regression, 31 days post dosing (FIG. 19). Alltreatment groups were well tolerated with minimal body weight loss forthe duration of the study.

(iii) NRAS-mutant melanoma model 21124

Patient derived “21124” (NRAS^(Q61H)) melanoma tumors were establishedin nude female mice. When tumors reached approximately 300 mm³, micewere randomized according to tumor volume into treatment groups (n=5).Treatment was initiated on Day 45 post xenograft implant. Anti-tumoractivity was determined on day 66 post tumor cell implantation.

Test agents were administered orally once daily (qd) or twice daily(bid) at the dose levels indicated in FIG. 20 at a dosing volume of 10ml/kg of animal body weight during course of treatment. Tumor volumeswere measured by digital caliper 2 times a week and body weights of allanimals were recorded throughout the course of treatment. Compound offormula (I) dosed at 100 mg/kg qd achieved 10% T/C, while trametinibdosed at 0.3 mg/kg qd achieved 54% T/C, 17 days post dosing. CombiningCompound of formula (I) dosed at 50 mg/kg bid with trametinib dosed at0.0375 mg/kg qd achieved 40% regression, 21 days post dosing (FIG. 20).All treatment groups were well tolerated with no body weight loss forthe duration of the study.)

(iv) NRAS-mutant melanoma model “20864”

Patient derived “21124” (NRAS^(Q61H)) melanoma tumors were establishedin nude female mice. When tumors reached approximately 300 mm³, micewere randomized according to tumor volume into treatment groups (n=5).Treatment was initiated on Day 19 post xenograft implant. Anti-tumoractivity of the single agents was determined on day 33 post tumorimplantation, while the anti-tumor activity of the combination betweenCompound of formula (I) and trametinib was determined on day 55 posttumor cell implantation.

Test agents were administered orally once daily (qd) or twice daily(bid) at the dose levels indicated in FIG. 21 at a dosing volume of 10ml/kg of animal body weight during course of treatment. Tumor volumeswere measured by digital caliper 2 times a week and body weights of allanimals were recorded throughout the course of treatment. Compound offormula (I) dosed at 50 mg/kg bid achieved 30% T/C, while trametinibdosed at 0.3 mg/kg qd achieved 84% T/C, 14 days post dosing. CombiningCompound of formula (I) dosed at 50 mg/kg bid with trametinib dosed at0.0375 mg/kg qd achieved 12% regression, 36 days post dosing (FIG. 21).On day 33, 2 mice in the combination group were sacrificed forpharmcodynamic studies, and the efficacy was carried with n=3 for theremainder of the study. All treatment groups were well tolerated withminimal body weight loss for the duration of the study.

Collectively, the data suggest that combined treatment with Compound offormula (I) and trametinib may achieve greater and more durableresponses in patients with activated MAPK pathway due togain-of-function mutations in the MAPK pathway. Some of the combinationdoses tested showed slight body weight loss (up to 10%,) however, themajority of combination doses tested. were well tolerated as judged byminimal body weight loss

Example 3 A Phase I Study of Compound of Formula (I) alone and inCombination with Trametinib in Adult Patients with Advanced Solid TumorsHarboring MAPK Pathway Alterations

The recommended starting dose and regimen of Compound of formula (I) assingle agent in this study is 100 mg once a day (QD) orally based on thepreclinical safety, tolerability data, pharmacokinetic (PK) and/orpharmacodynamic (PD) data in preclinical studies, as well as exploratoryhuman efficacious dose range projection in order to minimize exposure topotentially toxic drug levels while limiting the number of patients thatmight receive inactive doses.

Starting doses of 100 mg, 200 mg, 250 mg, 300 mg, or 400 mg areenvisioned; preliminary data suggesting a starting dose of 250 mg oncedaily (QD) may be effective on solid tumors. For maximum flexibility ofdosing, Compound of formula (I) may be prepared as 50 mg and/or 100 mgtablets for oral administration. The QD regimen has been demonstrated tobe efficacious and tolerated in preclinical studies. In Calu6xenografts, similar levels of efficacy were achieved with either QD orfractionated twice a day (BID) regimens, suggesting efficacy is relatedto overall exposure. The predicted human PK and the predicted half-life(˜9h), also suggest efficacious exposure can be achieved with QD dosing.

The dose escalation of Compound of formula (I) in combination with a MEKinhibitor, e.g., trametinib, will begin with a dosing regimen identifiedfor Compound of formula (I) as a single agent: the starting dose ofCompound of formula (I) may be lower than the single agent dose. Theselection of this dose thus should minimize exposure to potentiallytoxic drug levels while limiting the number of patients that mightreceive doses too low to provide good efficacy. MEK inhibitor, e.g.,trametinib will be administered at a flat dose of 2 mg which is thesingle agent recommended dosage.

In the dose expansion part, patients in the combination arm will betreated at the recommended dose and regimen for the drug combinationbased on the dose escalation data.

In the combination studies, a daily dose of 400 mg of Compound offormula (I) QD and a daily dose of 1 mg of trametinib or a daily dose of400 mg of Compound of formula (I) and a daily dose of 2 mg trametinibare envisaged. The daily dose of Compound of formula (I) may bepreferentially administered twice daily, whilst the daily dose oftrametinib administered once daily. Other doses such as in the Tablebelow may also be administered.

TABLE Dose levels of the combination of Compound of formula (I) andtrametinib Daily dose for Compound of formula (I)* Daily dose fortrametinib* 100 mg 0.5 mg, 1.0 mg, 1.5 mg or 2.0 mg 200 mg 0.5 mg, 1.0mg, 1.5 mg or 2.0 mg 400 mg 0.5 mg, 1.0 mg, 1.5 mg or 2.0 mg 800 mg 0.5mg, 1.0 mg, 1.5 mg or 2.0 mg 1200 mg  0.5 mg, 1.0 mg, 1.5 mg or 2.0 mg*Daily doses of either Compound of formula (I) or trametinib, or both,may be administered once or twice daily.

In addition, an efficacious dosage can be determined by monitoringbiomarkers indicative of MAP kinase pathway inhibition. In particular,DUSP6 (dual specificity phosphatase 6) is a known biomarker for thispathway, and in vivo levels of DUSP6 have been shown to drop in asubject receiving a dosage of Compound of formula (I) that is associatedwith efficacious plasma levels of Compound of formula (I). Thus, DUSP6may be used as a pharmacodynamics biomarker in subjects treated withCompound of formula (I), whether as a single agent or in combinationwith a MEK inhibitor.

Example 4N-(2-methyl-5′-morpholino-6′-((tetrahydro-2H-pyran-4-y0oxy)-[3,3′-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide

-   Compound of formula (II) is a morpholine-substituted biaryl compound    of the following structure

-   Compound of formula (II) is an inhibitor of B-Raf and C-Raf. The    compound is disclosed and its preparation described in example 131    in published PCT patent application WO2014/151616.

Example 4A

Compound of formula (II) is a type II inhibitor of both mutated andwild-type B-Raf and C-Raf.

TABLE 5 Half maximal inhibitory concentration (IC-50) of Compound offormula (II) on B-Raf, B-Raf V600E and C-Raf Kinase activity inhibitionIC₅₀ % of target binding at 1 Target [mM] mM BRAF 0.0015 99.7BRAF^(V600E) 0.001 99.9 CRAF 0.0004 99.8

Example 4B

Compound of formula (II) exhibits activity on numerous human cancer celllines that express mutations in the MAPK pathway as shown in thefollowing Table. Activity is especially strong on cell lines that harborat least one mutation in BRAF or RAS.

TABLE 6 Effect of Compound of formula (II) on proliferation in a panelof human cancer cell lines. Compound of Dabrafenib formula (II)B-Raf/Ras Cell line IC₅₀ [mM] IC₅₀ [mM] alteration A375 0.003 0.13 BRafMutant IPC-298 0.27 0.07 NRas Mutant Calu-6 23 1.4 KRas Mutant HCT116 170.98 KRas Mutant Different tumor cell lines were treated with dosetitrations of Compound of formula (II) for 5 days, and cellproliferation was determined using the CellTiter-Glo ™ luminescent cellviability assay.

Example 4C

The activity of Compound of formula (II) and dabrafenib in cell linesharboring BRAFV600, NRAS or KRAS mutations was compared (FIG. 7). InA375 cells, both dabrafenib and Compound of formula (II) inhibited MEKand ERK phosphorylation to near completion at 0.05 and 0.5 μM,respectively. In contrast, in the three cell lines harboring either aNRAS or KRAS mutation, dabrafenib treatment at 0.05 and 0.5 p,M led toan increase in MEK and ERK phosphorylation, and only at 5 μM showedmodest inhibition. In comparison, Compound of formula (II) showeddose-dependent inhibition of MEK and ERK phosphorylation withoutapparent pathway activation in all three RAS mutant models (minimalactivation of pMEK in IPC-298 and HCT116) (FIG. 7). The ability ofCompound of formula (II) to inhibit pathway signaling was comparable incells harboring different RAS mutations and those with the BRAF V600mutation, reaching near complete inhibition of pMEK and pERK at 0.5 μM.

The anti-proliferation activity of Compound of formula (II) anddabrafenib in these cell lines was also investigated (FIG. 8).Consistent with the signaling data, dabrafenib showed the most potentanti-proliferative activity in BRAF^(V600)E A375 cells (IC₅₀=0.003 μM),approximately 100× less potent in NRAS^(mut) IPC-298 cells (IC₅₀=0.27μM), and very little activity in the two KRAS^(mut) cell lines (Calu-6IC₅₀=23 μM, HCT116 IC₅₀=17 μM). In contrast, Compound of formula (II)showed less IC₅₀ dose shift in RAS^(mut) models compared to theBRAF^(mut) model and exhibited dose-dependent growth inhibition in allcell lines examined, with IC₅₀ values of 0.13 μM, 0.07 μM, 1.4 μM, and0.98 μM in A375, IPC-298, Calu-6 and HCT116, respectively.

A similar experiment with the Compound of Formula (I) also demonstratedthat the Compound of Formula (I) produced similar results (see Tablebelow).

BRAFV600E Wild type (WT) Dimer/ monomer RAF dimer 2^(nd) Monomer pERKIC50 site monomer inhibition [μM] pERK IC50 [μM] ratio Dabrafenib 0.0053 600 Compound of 0.059 0.078 1.3 formula (I)

These data demonstrate that Compound of formula (I) and Compound offormula (II) exhibit a mode of inhibition distinct from the class of RAFmonomer inhibitors. Their activity inhibiting both RAF monomers anddimers suggest it should be effective in treating tumors harboring BRAFor RAS mutations, in contrast to dabrafenib which is unlikely to beeffective in RAS-mutant tumors

Example 4D

Antiproliferative activity of Compound of formula (II) was analyzed in abroad panel of genetically characterized human cancer cell line models.Activity of Compound of formula (II) was compared with that ofdabrafenib in 357 cell lines with mutations in BRAF, KRAS, or NRAS, orwild-type for both BRAF and RAS (FIG. 9). Each data point represents theinhibitor IC50 value in a cell line following 3 days of treatment. Usingan inhibitor IC50 of 5 μM as a cut-off, the number of sensitive (IC50<5μM) and insensitive (IC50>5 μM) lines was indicated within each geneticgroup for Compound of formula (II) and dabrafenib. For example, Compoundof formula (II) shows anti-proliferative activity in cancer cell linesthat harbor non-V600 mutation with or without a co-occurring KRASmutation, including ovarian cancer cell line Hey-A8(KRAS^(G120D)/BRAF^(G464E), IC₅₀=0.63 μM), breast cancer cell lineMDA-MB-231 (KRAS^(G13D)/BRAF^(G464V), IC₅₀=3.4 μM), and lung cancer cellline NCI-H1666 (BRAF^(G466V), IC₅₀=3.97 μM). In comparison, the BRAFmonomer inhibitor dabrafenib had IC₅₀>30 μM in all three cell linemodels. Data were analyzed with a Fisher's exact test to evaluatewhether sensitivity of cell lines to the inhibitor treatment wassignificantly increased in the mutant group when compared to wild-typecell lines. For Compound of formula (II), cell lines harboring B-Raf,KRAS, or N-Ras mutations exhibited significantly increased sensitivityas compared to those that are wild-type, with p-values of 3.09×10⁻¹⁷,1.19×10⁻⁴, and 1.26×10⁻⁶, respectively. The odds ratios are 28.9, 4.6and 10.6, respectively. In comparison, higher sensitivity to dabrafenibwas only significant in cell lines harboring B-Raf mutations, but notwith KRAS or N-Ras mutations, with p-values of 1.9×10⁻¹⁵, 0.11, and 0.25respectively.

Example 5 Anti-Tumor Activity of Compound of Formula (II) in KRAS-MutantNSCLC Models

Both signaling inhibition and anti-tumor efficacy of Compound of formula(II) were investigated in the KRAS mutant Calu-6 model in vivo. Calu-6tumor xenografts were generated by implanting cells in 50% MatrigelTMsubcutaneously into the right flank of female nude mice (6-8 weeks old).Tumor-bearing mice were randomized into treatment groups and treatedwith a single oral dose of Compound of formula (II) across a wide doserange (from 10 to 200 mg/kg). Tumor tissues were then collected atmultiple time points post-dose to levels of phospho- and total MEK1/2using the MesoScale Discovery (MSD) platform or DUSP6 mRNA byquantitative PCR (qPCR). As shown in FIG. 10, treatment with Compound offormula (II) led to inhibition of MEK phosphorylation in adose-dependent manner both in degree and in duration. Compound offormula (II) at both 100 and 200 mg/kg was able to suppressphosphorylated MEK (pMEK) to greater than 50% for more than 16 hours.Subsequently, anti-tumor efficacy of Compound of formula (II) wasevaluated in the same tumor xenograft model (FIG. 11). Tumor bearinganimals were dosed with vehicle, Compound of formula (II) at 10, 30,100, or 200 mg/kg, administered orally every day (qd) for 19 days.Anti-tumor activity was determined by assessing percentage of tumorvolume in the treatment groups versus that in vehicle-treated (% T/C) orpercentage of tumor regression compared to the starting volume (%regression). Tumor volume and body weights were collected at the time ofrandomization and twice per week for the study duration. Tumor volumewas determined by measurement with calipers and calculated using amodified ellipsoid formula, where tumor volume (TV)(mm³)=[((1×w2)×3.14159))/6], where 1 is the longest axis of the tumorand w is perpendicular to 1. In line with pMEK inhibition, treatmentwith Compound of formula (II) resulted in dose dependent anti-tumoractivity starting from 30 mg/kg (FIG. 11). Treatment with 30 mg/kg ofCompound of formula (II) led to a 52% T/C, while treatment at 100 and200 mg/kg resulted in tumor regressions of 47% and 88%, respectively, inline with the more durable pathway inhibition at the two higher doselevels.

To assess the antitumor activity of Compound of formula (II) further, alarge scale in vivo screen of Compound of formula (II) efficacy in 23patient derived xenograft (PDX) models derived from patient withnon-small cell lung cancer (NSCLC) was performed (FIG. 12). Tumorresponse is presented as a waterfall plot of best average percentagechange in tumor volume with Compound of formula (II) treatment, andtumors were annotated for their mutation status of RAS or BRAF. Compoundof formula (II) dosed at 60 mg/kg or 200 mg/kg (▾) daily led to tumorgrowth inhibition in a subset of NSCLC PDX tumors, in which tumors thatharbor mutation of B-Raf, N-Ras, or KRAS, were enriched among the betterresponders. One of the B-Raf mutant tumors, HLUX1323, harbors a D594Nmutation which has been shown to activate signaling mediated through Rafdimerization and Compound of formula (II) led to 26% of tumor shrinkagein this model. These data further support the anticancer efficacy ofCompound of formula (II) in both Ras and B-Raf mutant cancer cells as aresult of its selective activity in inhibiting Raf monomers or dimersand the oncogenic MEK/ERK signaling.

Example 6 Synergistic Effect of Compound of Formula (II) with a MEKInhibitor

Growth inhibition of HPAF-II cells (KRAS mutant) was measured followingtreatment with Compound of formula (II) or trametinib as single agent,or with the two in combination, across a wide dose range. Isobologramsand synergy scores were generated to assess the combination activity. Asshown in FIG. 13, Compound of formula (II) in combination withtrametinib had a synergistic effect on inhibiting HPAF-II cell growth,with a Loewe synergy score of 11.1.

To evaluate the effect of the combination treatment on signalinginhibition versus either agent alone, nude mice bearing HPAF-IIxenograft tumors were treated with a single dose of Compound of formula(II) at 100 mg/kg, trametinib at 0.3 mg/kg or the two inhibitorscombined. DUSP6 mRNA levels, as a measurement of pathway activity, wasdetermined in tumor samples collected at multiple time points post-dose.As shown in FIG. 14, Compound of formula (II) treatment led to 83%inhibition of DUSP6 at 4 hours (hrs) post dose compared to vehiclecontrol, however, this inhibition was not durable as demonstrated by theincreased levels of DUSP6 at 16 and 24 hrs post dose. Similarly,trametinib treatment led to a partial and transient inhibition of DUSP6.In contrast, the combination of Compound of formula (II) and trametinibled to a more sustained DUSP6 inhibition, showing greater than 80% ofinhibition even at 16 hours post dose. Anti-tumor efficacy of thedifferent treatments in the same tumor xenograft model was evaluated:Tumor-bearing animals were dosed with vehicle, Compound of formula (II)at 100 mg/kg qd, trametinib at 0.3 mg/kg qd, or a combination of bothfor 10 days (FIG. 15). In line with DUSP6 inhibition, the combination ofCompound of formula (II) and trametinib treatment resulted in greateranti-tumor activity than either of the single agents alone, resulting in33% regression as compared to 40% T/C or 54% T/C by Compound of formula(II) or trametinib, respectively (FIG. 15). Collectively, these datasuggest that combined treatment with Compound of formula (II) andtrametinib achieves greater and more durable responses in patients withactivated MAPK pathway due to gain-of-function mutations in the MAPKpathway. It is also expected that Compound of formula (I) in combinationwith trametinib achieves greater and more durable responses than eithertreatment alone, resulting in enhanced anti-tumor activity in patientswith activated MAPK pathway due to gain-of-function mutations in theMAPK pathway.

INCORPORATION BY REFERENCE

All publications, patents, and Accession numbers mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication or patent was specifically and individually indicated to beincorporated by reference.

EQUIVALENTS

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

The invention claimed is:
 1. A pharmaceutical combination comprising aRaf inhibitor which is the Compound of formula (I)

or a pharmaceutically acceptable salt thereof, and (b) a MEK inhibitor,wherein the MEK inhibitor is trametinib, or a pharmaceuticallyacceptable salt or solvate thereof.
 2. The pharmaceutical combinationaccording to claim 1 wherein trametinib is in the form of a dimethylsulfoxide solvate.
 3. A method of treating cancer by treating a patientin need thereof with a pharmaceutical combination comprising a compoundof formula (I)

or a pharmaceutically acceptable salt thereof, and trametinib, or apharmaceutically acceptable salt or solvate thereof, wherein the canceris selected from the group consisting of KRAS-mutant NSCLC (non-smallcell lung cancer), NRAS-mutant melanoma, KRAS-mutant ovarian cancer andKRAS-mutant pancreatic cancer.
 4. The method according to claim 3wherein the cancer is N-RAS mutant melanoma.
 5. The method according toclaim 3 wherein the cancer is NSCLC.
 6. The method according to claim 5wherein the NSCLC is KRAS-mutant NSCLC, BRAF-mutant NSCLC, or bothKRAS-mutant and BRAF-mutant NSCLC.
 7. The method according to claim 5wherein the NSCLC is characterized by a BRAF V600E mutation or a BRAFnon-V600E mutation.
 8. The method according to claim 3, wherein thecancer is advanced or metastatic cancer.
 9. The method according toclaim 3 wherein the cancer is a BRAF V600-mutant melanoma or BRAFV600-mutant NSCLC, wherein the melanoma is responding to treatment or isno longer responding to treatment with a BRAF inhibitor, or a MEKinhibitor or both a BRAF and MEK inhibitor; wherein the BRAF inhibitoris selected from dabrafenib and vemurafenib; and the MEK inhibitor isselected from trametinib and cobimetinib.
 10. The method according toclaim 9 wherein the cancer is BRAF V600-mutant melanoma which isresistant to treatment with a combination of dabrafenib and trametinibor to treatment with a combination of vemurafenib and cobimetinib. 11.The method according to claim 9 wherein the cancer is BRAF V600-mutantNSCLC which is resistant to treatment with a combination of dabrafeniband trametinib.
 12. The method according to claim 3 wherein the cancerhas progressed following standard of care or for whom no effectivestandard therapy exists; wherein the cancer is selected from NSCLC andmelanoma.
 13. The method of claim 3 wherein the KRAS-mutant pancreaticcancer is KRAS-mutant pancreatic ductal adenocarcinoma (PDAC)).